Method of Treatment or Prevention of Hair Loss or for the Enhancement of Hair Growth

ABSTRACT

The present application relates to use of a Midkine family protein for growing hair on a mammal, or in the manufacture of a medicament for growing hair on a mammal, especially for treatment or prevention of different forms of alopecia.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of priority from AustralianApplication No. 2010900771 filed 24 Feb. 2010, the contents of which areincorporated in their entirety by reference.

FIELD OF THE INVENTION

The present application relates to the filed of therapy and preventionof hair loss or hair thinning in mammals, such as in humans sufferingfrom or having a propensity to develop alopecia, and to the promotion ofhair growth in mammals, such as in humans suffering from or having apropensity to develop alopecia.

BACKGROUND TO THE INVENTION Hair and Hair Development

Hair is integral to our body image and can have a profound influence onour self-esteem and self-confidence. The hair of non-human mammalspecies is commonly referred to as “fur”. Unless specifically statedotherwise, or the context requires otherwise, the term “hair” as usedherein shall be taken to include “fur”. The term “hair” shall also betaken to include hair on any part of a mammalian body, including theeyebrow, edge of the eyelid, armpit, and inside of the nostril, unlessthe context requires otherwise. Thus, hair may include head hair,eyebrow hair, eyelash, cilia, or other body hair.

Each hair comprises two structures: the shaft and the follicle. Theprimary component of the hair shaft is keratin. The hair shaft containsthree layers of keratin, however the inner layer i.e., the medulla, maynot be present. The middle layer i.e., the cortex, makes up the majorityof the hair shaft. The outer layer i.e., the cuticle, is formed bytightly-packed scales in an overlapping structure. Pigment cells aredistributed throughout the cortex and medulla giving the hair itscharacteristic color. The follicle contains several layers. At the baseof the follicle is a projection called a papilla, which containscapillaries, or tiny blood vessels, that feed the cells. The living partof the hair, the area surrounding the papilla called the bulb, is theonly part fed by the capillaries. The cells in the bulb divide every 23to 72 hours, faster than any other cells in the body. The follicle issurrounded by an inner root sheath and an outer root sheath. These twosheaths protect and mould the growing hair shaft. The inner root sheathfollows the hair shaft and ends below the opening of a sebaceous (oil)gland, which produces sebum, and sometimes an apocrine (scent) gland.The outer root sheath continues all the way up to the sebaceous gland.An erector pili muscle attaches below the sebaceous gland to a fibrouslayer around the outer sheath. When this muscle contracts; it causes thehair to stand up.

Human skin comprises two types of hair: vellus hair and terminal hair.Vellus hair is short, fine, “peach fuzz” body hair. It is a very soft,generally pale, and short hair that grows in most places on the humanbody in both sexes. Vellus hair is generally less than two centimetresin length, and the follicles from which vellus hair grows are notconnected to sebaceous glands. It is observed most easily in thosehaving less terminal hair to obscure it, such as women and children, Italso is found in pre-adolescents and in males exhibiting male-patternbaldness. Terminal or “androgenic” hair is developed hair, which isgenerally longer, coarser, thicker and darker than vellus hair. Phasesof growth in terminal hair are also more apparent than in vellus hair,by virtue of a generally-longer anagen phase. Terminal hair hasassociated sebaceous glands. In puberty, some vellus hair may developinto terminal hair. Under other conditions, such as male patternbaldness, terminal hair may revert to a vellus-like state

There are three sequential stages of hair growth: catagen, telogen, andanagen. Anagen is the active growth phase of the hair during which thecells in the root of the hair are dividing rapidly. Anagen hairs areanchored deeply into the subcutaneous fat and cannot be pulled outeasily. When a new hair is formed, it pushes the club hair up thefollicle, and eventually out. During this phase, the hair grows about 1cm every 28 days. Scalp hair stays in this active phase of growth for2-6 years. Human subjects that have difficulty growing their hair beyonda certain length may have a shortened anagen phase, whereas those havingan ability to grow longer hair quickly may have a longer anagen phase.In humans, the hair on the arms, legs, eyelashes, and eyebrows generallyhas a short anagen compared to head or scalp hair. The catagen phase isa transitional stage that lasts for about 2-3 weeks in humans, duringwhich time growth stops, thereby forming “club” hair. Telogen is aresting phase, lasting for about 100 days for scalp hair and much longerfor other body hair. During telogen, the hair follicle is at rest, theclub hair is formed, and compared to hair in anagen, the hair in telogenis located higher in the skin and can be pulled out readily. The root oftelogen hair comprises a visible solid, hard, dry, and white material.Shedding of telogen hair is normal, and up to 75 hairs in telogen areshed from the human scalp daily. The shed hairs are normally replaced asabout the same number of follicles enter anagen daily. At any time innormal scalp, approximately 80% to 90% of follicles are in anagen, about1% to 3% are in catagen i.e., undergoing involution, and about 5% to 10%are in telogen.

Conditions of Hair Loss and/or Reduced Hair Growth

Hair loss or hair thinning includes any condition that results in areduced ability to replace shed hairs or that results in enhancedshedding without their concomitant or subsequent replacement e.g.,brittle hair growth, thin hair growth, short hair growth, sparse hairgrowth, alopecia, or hair de-pigmentation. For example, the hair cyclecan become uncontrolled leading to accelerated hair loss, which may betemporary or permanent. As used herein, the term “alopecia” is used torefer to hair loss, unless specifically stated otherwise or the contextrequires otherwise.

Alopecia can have various causes. Hereditary androgenic alopecia is thecommonest form of alopecia: it is manifested by a decrease in hairvolume, or even baldness, and affects up to about 70% of men. Acutealopecia may be associated with treatment by chemotherapy, stress,severe malnutrition, iron deficiency, hormonal disorders, AIDS, or acuteirradiation. Alopecia areata, which seems to be of auto-immune origin(mechanism of cellular mediation), is characterized by “patches” ofvarying size in one or more body places. Alopecia totalis refers to aform of alopecia areata that extends over the entire scalp, and alopeciauniversalis refers to a form of alopecia areata that extending over thewhole body. Mechanistically, in all forms of alopecia, hair loss isdirectly-related to a reduced ability, slowing or failure of thefollicle to enter the anagen phase, or a failure to maintain thefollicle in the anagen phase, such that formation of a hair shaftreduces, is slowed or ceases altogether. Hair may move into the catagenphase before sufficient growth is achieved in the anagen phase, thusbecoming in a sustained manner short and thin (i.e. “hair thinning”).Chemotherapeutic agents, radiotherapeutic agents, and other medicinalproducts may induce necrosis or apoptosis of the follicle as aside-effect of the therapy, also preventing the follicle to enteranagen. For example, alkylating agents e.g., temozolomide, busulfan,ifosamide, melphalan hydrochloride, carmustine, lomustine orcyclophosphamide, and antimetabolites e.g., 5-fluorouracil,capecitabine, gemcitabine, floxuridine, decitabine, mercaptopurine,pemetrexed disodium, methotrexate or dacarbazine, and natural productse.g., vincristine, vinblastine, vinorelbine tartrate, paclitaxel,docetaxel, ixabepilone, daunorubicin, epirubicin, doxorubicin,idarubicin, mitoxantrone, mitomycin, dactinomycin, irinotecan,topotecan, etoposide, teniposide, etoposide phosphate, or bleomycinsulfate, and biologics e.g., filgrastim, pegfilgrastim, bevacizumab,sargramostim or panitumumab, and hormones or hormone-related agentse.g., megestrol acetate, fluoxymesterone, leuprolide, octreotideacetate, tamoxifen citrate or fluxymesterone, and other therapeuticagents e.g., sorafenib, erlotinib, oxaliplatin, dexrazoxane, anagrelide,isotretinoin, bexarotene, vorinostat, adriamycin, cytoxan, taxol,leucovorin, oxaliplatin, and combinations of the foregoing agents areknown to induce temporary or permanent alopecia.

Alopecia caused by any of the foregoing factors is a source of lowself-esteem and anxiety for many patients. For those undergoingchemotherapy or radiation therapy for cancer, alopecia adds todiscomfort from other adverse side-effects of the therapy e.g., nausea,skin rash, etc. Many alopecia sufferers, including patients receivingchemotherapy, choose to use wigs, hair pieces, scarves, hats or turbansto cover their bald or thinning regions. Those suffering from hair lossoften experience embarrassment and fear being ridiculed by othersbecause they look different. Some may take to wearing oversizedeyeglasses in an attempt to hide the absence of eyelashes and/oreyebrows. In some subjects, alopecia may lead to depression.

Animal Models of Alopecia

There are several useful models of alopecia in humans, that have beenacknowledged in the art for use in testing efficacy of alopecia remediesand other hair growth-promoting therapies.

For example, the stumptailed macaque possesses hereditary baldingcharacteristics similar in many respects to that of androgenic alopeciain humans, is used to obtain a morphometric assessment of the rate ofcyclic change of the hair follicle, including rates of cyclicprogression (resting to regrowing phase, and regrowing to late anagenphase) and overall changes in follicular size. These primates are alsoreasonably good predictors of compound efficacy, and for example, havebeen employed to test efficacy of minoxidil on androgenic alopecia.Cessation of topical minoxidil treatment resulted in a renewal of thebalding process, with folliculograms demonstrating increases in theproportion of resting follicles. This withdrawal from treatmentapparently had no effect on hair regrowth during subsequentreapplications of minoxidil. Such treatment resulted in regrowth similarto that in the first treatment phase. Continuous treatment of topicalminoxidil for 4 years has not resulted in systemic or local side effectsin these animals. See e.g., Brigham et al., Clin. Dermatol. 6,177-187,1998; Sundberg et al., Exp. Mol. Pathol. 67, 118-130 (1999), thecontents of which are incorporated herein by reference in theirentirety).

Collectively, the findings obtained from studies on mouse models supportthe concept of alopecia areata as an autoimmune disease, and severalrodent models with spontaneous and induced alopecia areata have beenidentified. For example, the Dundee Experimental Bald Rat (DEBR) was thefirst rodent model validated that developed spontaneous alopecia areataand is utilized to identify candidate alopecia areata susceptibilitygene loci (Michie et al., Br J Dermatol., 125, 94-100, 1991,incorporated herein by reference). The most extensively-characterizedand readily-accessible alopecia areata model is the C3H/HeJ mouse model(Sundberg et al., J Invest Dermatol., 102, 847-56, 1994, incorporatedherein by reference). Aging C3H/HeJ mice (females at 3-5 months of ageor older and males at more than 6 months of age) develophistopathological and immunohistochemical features of human alopeciaareata. Alopecia develops diffusely or in circular areas on the dorsalsurface of sufficiently-aged animals. Histologically, the changes inthis non-scarring alopecia appear limited to anagen follicles surroundedby mononuclear cells composed primarily of cytotoxic or cytostatic(CD8+) and helper (CD4+) T cells, this is associated with follicular andhair shaft dystrophy. Pedigree tracing of affected C3H/HeJ mice suggeststhat this non-scarring alopecia may be an inherited and complexpolygenic disease with a female predominance at younger ages. C3H/HeJmice with alopecia areata can be used to study the efficacy of currenttreatments of alopecia areata, to study the effectiveness and safetyprofile of new treatment forms in established alopecia areata, and toassess the influence of various factors on the development of alopeciaareata in order to prevent the onset of the disease.

Paus et al., Am. J. Pathol. 144, 719-734 (1994) have also described arodent model of acute alopecia, the entire content of which isincorporated herein by reference. In this model, alopecia is induced bya single intraperitoneal injection of cyclophosphamide to C57 BL/6 mice.In depilated C57 BL/6 mice, the hair follicles are synchronized toanagen. By day 9 after depilation, all follicles are mature anagen VIfollicles, and the skin is characterized by grey-to-black coloured hairshafts. Histologically, macroscopically, and functionally,depilation-induced anagen VI follicles are indistinguishable fromspontaneously-developing anagen follicles. Around day 16 afterdepilation, follicle regression occurs without loss of hair shafts inthe depilated animals, and skin colour converts from black to pink,indicating both induction of catagen and cessation of melanogenesis. Thedevelopment of catagen follicles is indicated macroscopically by achange in skin color from black to light grey, and occurs in large wavesappearing in the neck region first and then the flanks and tail regions.At day 20 after depilation, all follicles enter telogen again,characterized by change in skin color from grey to pink. Whencyclophosphamide is administered to C57 BL/6 mice on day 9 afterdepilation, the animals show rapid and reproducible visible signs ofacute alopecia dose-dependent, including significant loss of fur andpremature termination of anagen characterized by depigmentation leadingto a grey skin appearance by day 12-14. Thus, follicles of the neckregion are generally in catagen 3-5 days after cyclophosphamidetreatment. Hair shafts on the backs of animals are also removed easilyby rubbing at days 12-14, and by day 15, as much as 60% of the dorsalsurface may be exhibit alopecia. The color change and alopecia inducedby cyclophosphamide reflect the induction of dystrophic forms of anagenand catagen in anagen VI follicles. In cyclophosphamide-treated animals,follicles also progress to telogen rapidly, as evidenced by pink skin,and rapid loss of fur due to damage of the hair follicle. Telogen isshortened following cyclophosphamide treatment, and normal telogen hairfollicles enter the next hair cycle, so that animals develop new hairshafts on days 16-20 i.e., within about 7-10 days following treatment.These new hair shafts are often de-pigmented due to the presence ofdystrophic anagen follicles that have not had time to produce new,normally-pigmented hair shafts. Later, pigmented hair shafts develop.

Therapy for Conditions of Hair Loss and/or Reduced Hair Growth

Existing therapies for alopecia include topical minoxidil andderivatives thereof e.g., U.S. Pat. Nos. 4,139,619 and 4,596,812, andEuropean Pat. Nos. EP-0353123, EP-20 0356271, EP-0408442, EP-0522964,EP-0420707, EP-0459890 and EP-0519819, spironolactone, cyproteroneacetate, flutamide, finasteride, progesterone or estrogen. Anti-androgenagents such as finasteride and minoxidil are known for treatingandrogenic alopecia. None of these treatments is broadly applicable. Forexample, such treatments may not prevent hair loss during treatment witha chemotherapeutic agent. On the other hand, such compounds may produceundesirable side-effects. For example, minoxidil is a potentvasodilator. Patients may also require frequent dosing with suchcompounds to achieve an effective outcome. For example, minoxidilprovides very transient effects, because cessation of topical minoxidiltreatment results generally in a renewal of the balding process, withfolliculograms demonstrating increases in the proportion of restingfollicles. Minoxidil is also recommended for administration twice-dailyat 2% concentration. Notwithstanding that finasteride provides anadvance over minoxidil in being deliverable orally, and is considered tobe the best treatment available, about 35% or more of balding malerecipients show poor or no response to that drug. Finasteride may alsoproduce significant side-effects for some users, as a number of maleusers have reported erectile dysfunction, impotence, low libido, orgynecomestica after using that drug. In those males suffering suchside-effects, the side effects may not disappear after ceasingfinasteride.

Various prostaglandin analogs have also been disclosed for use intreatment of androgenic alopecia e.g., travoprost, voprostol, and thesemay also require frequent administration e.g., at least daily, howeversingle dosages of travoprost have been described e.g., U.S. Pat.Publication 20100190853. Prostaglandin analogs are also known for use intreatment of alopecia associated with chemotherapy e.g., U.S. Pat.Publication 20110002286. Prostaglandin analogs may have a variety ofadverse effects e.g., muscular constriction mediating inflammation,calcium movement, hormone regulation and cell growth control.

Midkine Family Proteins

Midkine (MK) is a growth/differentiation factor that was firstdiscovered as a gene product expressed transiently in the process ofdifferentiation induction of embryonic tumor cells (EC) with retinoicacid. MK is known to produce a broad range of adverse and beneficialbiological effects. The expression of MK is increased in human cancercells, including esophageal cancer, thyroid cancer, urinary bladdercancer, colorectal cancer, gastric cancer, pancreatic cancer, chestcancer, liver cancer, lung cancer, breast cancer, neuroblastoma,neuroblastoma, glioblastoma, uterine cancer, ovarian cancer, and Wilms'tumor, and is believed to promote the survival and migration of cancercells and to facilitate neovascularization. MK is also known to promotethe migration of inflammatory cells such as macrophages and neutrophil,leading to inflammation. MK is also known to stimulate the proliferationof cultured endometrial interstitial cells during endometriosis. Thus,MK inhibitors e.g., antibodies, aptamers or RNAi targeting MK protein orRNA, have been disclosed for use in treatment of a broad range ofinflammatory diseases such as arthritis, autoimmune disease, rheumaticarthritis, osteoarthritis, multiple sclerosis, postoperative adhesion,inflammatory colitis, psoriasis, lupus, asthma, neutrophil functionalabnormalities, and endometriosis.

Beneficial effects of MK are also known, wherein MK is also involved inpromoting the formation of nascent intima following blood vessel damageand the onset of nephritis in an ischemic event, and in reducingpostoperative adhesions in rheumatic subjects. Thus, MK protein has beendisclosed for use in treatment of cerebral ischemia, cardiac ischemia,restenosis following vascular reconstruction surgery, cardiac coronaryarterial vascular obstructive disease, cerebral vascular obstructivedisease, renal vascular obstructive disease, peripheral vascularobstructive disease, and arteriosclerosis.

Pleiotrophin (PTN or HB-GAM) is a midkine family protein havingapproximately 50% identity at the amino acid sequence level to MK. BothMK and PTN comprise a high content of cysteine and basic residues. Allthe 10 cysteine residues are conserved in MK and PTN, and structurally,both can be divided into the N-domain and the C-domain. As a result ofNMR analysis, it is known that these two molecules have very similarthree-dimensional structures. Each domain consists of three f3 sheets,connected via a flexible linker region. K79, R81, and K102, consideredto be important to the binding of to chondroitin sulfate and heparin,are conserved between the two proteins. MK and PTN also sharethree-dimensional structures wherein these basic residues appear in thevicinity of the protein surface. Accordingly, PTN has been disclosedpreviously for the same medical indications as MK.

It is to be understood that, if any prior art publication is referred toherein, such reference does not constitute an admission that thepublication forms a part of the common general knowledge or backgroundart in Australia or elsewhere.

SUMMARY OF THE INVENTION

In work leading up to the present invention, the inventor sought todevelop protein-based reagents, e.g., for topical administration to asubject, that enhance hair growth or the initiation of hair growth insubjects that are susceptible to hair loss or suffer hair loss. Theinventor also sought to develop an effective topical protein-basedformulation that can be applied readily to the skin and exert an effectnotwithstanding the barrier that the dermis generally provides toexternal agents.

The inventor developed an animal model of hair loss consistingessentially of an aged mouse that suffers hair loss e.g., in the form ofbald regions or alopecic sites. The inventor found that topicalapplication of midkine or pleotrophin protein to the animal modelinduced hair growth or the recommencement of hair growth in these baldregions or alopecic sites. Topical formulations of midkine orpleiotrophin were found to be superior to parenteral formulations inproducing these hair growth promoting effects in this animal model.

The data provided herein also show that, in contrast to minoxidil,midkine and pleiotrophin failed to stimulate hair growth visibly inhealthy animals that have been shaved to synchronize their follicle inanagen, and exhibited no clinical symptoms of alopecia. This suggeststhat midkine and pleiotrophin may act by a more specific mechanism thanminoxidil that does not comprise a vasodilatory effect of the dermalpapillae. Thus, fur regrowth stimulated by a midkine family protein mayarise from an anti-apoptotic effect of midkine on follicles to therebyextend their anagen phase, or alternatively, or the re-entry of normaltelogen follicles into the next hair cycle.

In further work leading to the present invention, the applicant utilizedthe accepted animal model of chemotherapy-induced alopecia (CIA)described e.g., by Paus et al., Am. J. Pathol. 144, 719-734 (1994), thecontents of which are incorporated herein in their entirety. Theapplicant performed a randomised trial in which all animals were shavedand waxed, and five groups of animals were administered daily with atopical formulation of midkine protein for 7 days before and 19 daysfollowing i/p injection of cyclophosphamide, and another two groups ofanimals were administered daily with a topical formulation of midkineprotein for 19 days following i/p injection of cyclophosphamide. Controlmice received a saline placebo. Data presented herein indicate thattopical administration of a midkine family protein e.g., MK and/or PTN,before chemotherapy, or before and after chemotherapy, significantlyreduces the severity of fur loss i.e., promotes fur growth at all timepoints following administration of the chemotherapeutic agent. Treatmentwith a midkine family protein also retains a greater proportion offollicles in anagen following treatment with cyclophosphamide, asevidenced by more animals having black or dark grey skin when treatedwith midkine family protein than for the group receiving a placebo. Thissuggests an anti-apoptotic effect on anagen follicles. These beneficialeffects are detectable in the period immediate following chemotherapywhen follicles have not yet recovered, and during later folliclerecovery after chemotherapy has ceased. Data presented herein alsoindicate that topical administration of a midkine family protein e.g.,MK and/or PTN, following chemotherapy at least improves recovery offollicles, as demonstrated by a reduced incidence of alopecia comparedto subjects receiving a placebo. Thus, animals treated daily withtopical midkine protein following chemotherapy also lose less hair intotal than animals not receiving treatment, and the reduced hair loss isnearly as effective as in animals treated with midkine protein beforeand after chemotherapy. Subjects receiving midkine therapy before orafter chemotherapy also exhibit a higher rate of hair growth during thefollicle recovery period, suggesting an anti-apoptotic effect of midkineon follicles to thereby extend their anagen phase, or alternatively,enhanced re-entry of normal telogen follicles into the next hair cyclefollowing treatment with midkine.

It is to be understood that the methods described herein for the agedmouse model of hair loss are transferable to testing in other animalmodels of alopecia of humans, including the stump-tailed macaque 21-23model of androgenic alopecia and/or the hairless (hr/hr) model ofpapular atrichia, as described herein.

The data provided herein therefore support the use of compositionscomprising a midkine family protein in promoting hair growth and/orreducing hair loss and/or reducing hair thinning and/or preventing hairloss and/or preventing hair thinning and/or delaying hair loss and/ordelaying hair thinning e.g., in aging subjects, subjects suffering fromalopecia such as androgenic alopecia or acute alopecia. Typically,effective compositions f the invention for treatment and/or preventionof alopecia are formulated for topical administration, howeverparenteral formulations may also be employed, such as forco-administration with a cytotoxic or cytostatic agent of acute alopeciathat is gendrally administered parenterally e.g., a chemotherapeuticagent.

Accordingly, one example of the present invention provides a formulatione.g., a topical formulation, comprising an amount of a midkine familyprotein, such as midkine, pleiotrophin or a variant or homolog ofmidkine or pleiotrophin, and a topical carrier, excipient or emollient,effective to prevent hair loss and/or promote hair growth and/or enhancehair growth. By “topical formulation” is meant that the formulation iscapable of being applied externally to the dermis of a mammal, or isapplied to the dermis. For example, the formulation may be useful foradministration to the dermis or skin of a male subject who issusceptible to hair loss or at risk of hair loss, such as a subjectsuffering from or at risk of developing male-pattern baldness. Inanother example, the formulation of the invention is useful foradministration to the dermis or skin of a subject suffering from adisease or condition associated with hair loss e.g., alopecia,especially acute alopecia or androgenic alopecia.

In one example, the topical formulation comprises an amount of a midkinefamily protein and a topical carrier, excipient or emollient effectiveto treat or prevent hair loss and/or promote hair growth and/or enhancehair growth. Preferably, the formulation is for administration to thedermis or skin of a subject who is susceptible to hair loss or at riskof hair loss, or for administration to the dermis or skin of a subjectsuffering from hair loss. It will be appreciated that the topicalformulation is suitable for treatment or prevention of alopecia e.g., anacute form of alopecia or androgenic alopecia. For example, theformulation may be used for treatment or prevention of alopecia in asubject undergoing treatment with a cytotoxic agent or cytostatic agentor to whom treatment with a cytotoxic agent or cytostatic agent has beenprescribed. The midkine family protein may be midkine, pleiotrophin,midkine-like protein, or a truncated midkine protein.

The present invention thus provides improved topical formulationscomprising a therapeutically-effective amount of a midkine familyprotein e.g., an effective amount of a midkine protein or pleiotrophinprotein or biologically-active variant thereof to enhance hair growth.The topical formulations of the invention are particularly useful forthe prevention or treatment of alopecia, including androgenic alopecia,acute alopecia, alopecia areata, alopecia totalis and alopeciauniversalis. The topical formulations of the invention are preferablyfor treatment of androgenic alopecia. The topical formulations of theinvention are even more preferably for the treatment of acute alopeciain subjects undergoing therapy with a cytotoxic or cytostatic compoundthat causes hair loss, especially a chemotherapeutic agent,radiotherapeutic agent, or antiviral compound such as administered to asubject infected with HIV-1. The topical formulations of the inventionare also preferably for the prevention of acute alopecia in subjectsabout to undergo therapy with a cytotoxic or cytostatic compound thatcauses hair loss, especially a chemotherapeutic agent, radiotherapeuticagent, or antiviral compound such as administered to a subject infectedwith HIV-1.

It is to be understood that the topical formulations of the inventionare useful for the stimulation of hair growth in subjects suffering fromor having a predisposition to develop androgenic alopecia or malepattern baldness, and/or in subjects about to undergo therapy with acytotoxic or cytostatic compound that causes hair loss, or undergoingsuch therapy.

It is also to be understood that the topical formulations of the presentinvention are compatible with various types of therapeutic agents orcarriers with which they are combinable or capable of being administeredsequentially or simultaneously or concomitantly. For example a topicalformulation comprising a midkine family protein may further comprise asecond compound for treatment of the same condition and/or beco-administered with such second compound(s). In such circumstances, theefficacy of the midkine family protein is supplemented by the action ofthe second compound. For example, the midkine family protein isco-administered with cestradiol and/or oxandrolone and/or minoxidiland/or finasteride or an agent that blocks the conversion oftestosterone to dihydrotesterone. Alternatively, or in addition, thetopical formulation of the invention is co-administered with a cytotoxicor cytostatic compound that causes hair loss e.g., in the case of asubject undergoing chemotherapy or radiation therapy or treatment forHIV-1 infection or AIDS. such circumstances, the efficacy of the midkinefamily protein counteracts the hair-loss effect of the cytotoxic orcytostatic compound.

Another example of the present invention provides for the use of amidkine family protein in the manufacture of a medicament e.g., atopical medicament for preventing and/or treating hair loss in a subjectsuffering from alopecia or having a propensity to develop alopecia. Themedicament may be for administration to a section of the population thatis susceptible to hair loss or suffers from hair loss, such as a subjectsuffering from or at risk of developing male-pattern baldness. Forexample, the subject may have no visible symptoms of alopecia, howeversuffers from a genetic condition that predisposes the Subject toalopecia, or the subject may be about to undergo therapy with acytotoxic or cytostatic agent or antiviral compound that inducesalopecia. In another example, the topical medicament is useful foradministration to the dermis or skin of a subject suffering from adisease or condition associated with hair loss e.g., alopecia. Forexample, the subject may have a pre-existing alopecia or be undergoingtherapy with a cytotoxic or cytostatic agent or antiviral compound thatinduces alopecia whether or not visible symptoms of alopecia havedeveloped before midkine family protein therapy is commenced. Themidkine family protein is especially useful in manufacture of amedicament for treatment or prevention of acute alopecia in a subjectundergoing chemotherapy, and such a medicament may be formulated foradministration by a topical or parenteral route, however topicalformulations are preferred.

By “topical medicament” is meant that the active agent i.e., midkinefamily protein, such as midkine, pleiotrophin or a variant or homolog ofmidkine or pleiotrophin, is formulated with a topical carrier, excipientor emollient for application to the dermis of a mammal.

In one particularly preferred example, the use of a midkine familyprotein is in the manufacture of a medicament for treatment orprevention of alopecia, e.g., an acute form of alopecia or androgenicalopecia, in a human or other mammalian subject. The subject may be ahuman or mammalian subject a subject undergoing treatment with acytotoxic agent or cytostatic agent or to whom treatment with acytotoxic agent or cytostatic agent has been prescribed. The medicamentmay be a formulation for topical application. The midkine family proteinmay be midkine, pleiotrophin, midkine-like protein, or a truncatedmidkine protein.

Another example of the present invention provides a method of treatmentor prevention of hair loss, said method comprising administering to asubject in need thereof e.g., a subject suffering from alopecia orhaving a propensity to develop alopecia, a formulation, e.g., a topicalformulation, comprising an amount of a midkine family protein, such asmidkine, pleiotrophin or a variant or homolog of midkine orpleiotrophin, and a topical carrier, excipient or emollient, effectiveto prevent hair loss and/or promote hair growth and/or enhance hairgrowth on the subject. The subject will generally be a mammal such as ahuman. For example, the method of the invention is useful for thetreatment of male subjects who are susceptible to or at risk of hairloss such as in male-pattern baldness. Such subjects may have no visiblesymptoms of alopecia, however suffers from a genetic condition thatpredisposes the subject to alopecia, or the subject may be about toundergo therapy or be undergoing therapy with a cytotoxic or cytostaticagent or antiviral compound, that induces alopecia. In another example,the method of the invention is useful for the treatment of a subjectsuffering from a disease or condition associated with hair loss e.g.,alopecia. Such subjects may have a pre-existing alopecia or beundergoing therapy with a cytotoxic or cytostatic agent or antiviralcompound that induces alopecia wherein symptoms of alopecia havedeveloped. The treatment and prevention of subjects undergoingchemotherapy or to whom chemotherapy has been prescribed is clearlyencompassed by the inventive method.

Another example of the present invention provides a method of promotingor enhancing hair growth or hair initiation, such as during or followingtherapy with a cytotoxic or cytostatic agent or antiviral compound thatinduces alopecia, said method comprising administering to a subject inneed thereof a formulation e.g., a topical formulation comprising anamount of a midkine family protein, such as midkine, pleiotrophin or avariant or homolog of midkine or pleiotrophin, and a topical carrier,excipient or emollient, effective to prevent hair loss and/or promotehair growth and/or enhance hair growth on the subject.

In another example, the present invention provides a method of treatmentor prevention of hair loss, said method comprising administering to asubject in need thereof, e.g., a subject suffering from alopecia such asan acute form of alopecia or androgenic alopecia or susceptible todeveloping said alopecia, a formulation comprising an amount of amidkine family protein and a carrier, excipient or emollient, effectiveto prevent hair loss and/or promote hair growth and/or enhance hairgrowth on the subject. Preferably, the formulation is a topicalformulation and said method comprises administering the topicalformulation to an affected area of the skin of the subject in which hairhas been lost or to an area of skin from which hair is likely to belost. The formulation may be administered to a subject suffering fromalopecia for a time and under conditions sufficient to reduce hair lossand/or effect hair growth in the subject. Alternatively, or in addition,formulation may be administered to a subject suffering from alopecia fora time and under conditions sufficient to prevent or reduce apoptosis offollicles of the subject. Alternatively, or in addition, formulation maybe administered to a subject suffering from alopecia for a time andunder conditions sufficient to extend an anagen phase of follicles ofthe subject. Alternatively, or in addition, formulation may beadministered to a subject suffering from alopecia for a time and underconditions sufficient to promote or advance entry of normal telogenfollicles of the subject into a following hair cycle. The methodaccording to the invention is particularly useful for subjectsundergoing treatment with a cytotoxic agent or cytostatic agent or towhom treatment with a cytotoxic agent or cytostatic agent has beenprescribed. In a preferred example, the midkine family protein may bemidkine, pleiotrophin, midkine-like protein, or a truncated midkineprotein.

In another example, the invention provides a method of promoting orenhancing hair growth or hair initiation in a subject suffering fromhair loss, said method comprising administering to the subject a topicalformulation comprising an amount of a midkine family protein and atopical carrier, excipient or emollient effective to prevent hair lossand/or promote hair growth and/or enhance hair growth on the subject.Preferably, the midkine family protein is midkine or pleiotrophin.

In another example, the invention provides a method of reducing hairloss in a subject undergoing chemotherapy or to whom chemotherapy hasbeen prescribed, said method comprising administering to the subject aformulation comprising an amount of a midkine family protein and acarrier, excipient or emollient for a time and under conditionssufficient to prevent or reduce hair loss due to the chemotherapy.Preferably, the method comprises administering the formulation topicallyto a subject that has been treated with the chemotherapy or to whomchemotherapy has been prescribed before commencement of thechemotherapy. In one example, the method comprises administering theformulation topically to a subject to whom chemotherapy has beenprescribed before and after commencement of the chemotherapy. Asexemplified herein, the midkine family protein may be midkine.

Another example of the present invention provides an animal model ofhair loss consisting essentially of an aged mouse that suffers hair losse.g., in the form of bald regions or alopecic sites. The presentinvention clearly extends to the use of this animal model to screen fornovel therapeutic and prophylactic compositions of matter that preventhair loss and/or promote hair growth and/or enhance hair growth, whereinsaid screen comprises administering a test compound to the animal anddetermining hair growth and/or reduced hair loss and wherein reducedhair loss and/or increased hair growth and/or an initiation of hairgrowth on the animal in the presence of the test compound relative to inthe absence of the test compound is indicative of the test compoundpreventing hair loss and/or promoting hair growth and/or enhancing hairgrowth. In one example of this screening method, the activity of thetest compound is compared to the activity of a midkine family proteinand a test compound having comparable or enhanced activity relative tothe midkine family protein is identified and/or isolated and/orformulated for topical administration in a method of the inventionaccording to any example hereof.

In another example, the present invention provides a method ofidentifying or isolating a compound that prevents hair loss and/orpromotes hair growth and/or enhances hair growth, wherein said methodcomprises administering a test compound to a mouse, said mouse beingaged more than one year and suffering hair loss and having a knockout ofone allele of a gene encoding a midkine protein and one other functionalallele of the gene, and then determining hair growth and/or reduced hairloss on the mouse, and wherein reduced hair loss and/or increased hairgrowth and/or an initiation of hair growth on the mouse in the presenceof the test compound relative to in the absence of the test compound isindicative of the test compound preventing hair loss and/or promotinghair growth and/or enhancing hair growth.

Definitions

This specification contains nucleotide and amino acid sequenceinformation prepared using PatentIn Version 3.6, presented herein afterthe claims. Each nucleotide sequence is identified in the sequencelisting by the numeric indicator <210> followed by the sequenceidentifier (e.g. <210>1, <210>2, <210>3, etc). The length and type ofsequence (DNA, protein (PRT), etc), and source organism for eachnucleotide sequence, are indicated by information provided in thenumeric indicator fields <211>, <212> and <213>, respectively.Nucleotide sequences referred to in the specification are defined by theterm “SEQ ID NO:” followed by the sequence identifier (e.g. SEQ ID NO: 1refers to the sequence in the sequence listing designated as <400>1).

As used herein the term “derived from” shall be taken to indicate that aspecified integer may be obtained from a particular source albeit notnecessarily directly from that source.

Throughout this specification, unless the context requires otherwise,the word “comprise”, or variations such as “comprises” or “comprising”,is understood to imply the inclusion of a stated step or element orinteger or group of steps or elements or integers but not the exclusionof any other step or element or integer or group of elements orintegers.

The term “hair” means any hair or fur on the body of a mammal includinga human, and includes, for example, head hair, eyebrows, eyelashes,moustaches, beards, chest hair, back hair, arm hair, leg hair, genitalhair, nasal hair or ear hair.

As used herein, the term “treat” or “treating” or “treatment” shall betaken to include therapeutic treatment of a pre-existing condition, orprophylactic or preventative measures, wherein the aim is to prevent,ameliorate, reduce or slow down (lessen) hair thinning, hair loss oralopecia. It follows that hair growth, or treatment of hair thinning,refers to normalization of thinned hair, such as in alopecia, and/orincreasing the length and thickness of hair. A mammal in need oftreatment may already have the condition, or may be prone to have thecondition or may be in whom the condition is to be prevented. Suchtreatment preferably involves an anti-apoptotic effect on follicles tothereby extend their anagen phase, or alternatively, or the re-entry ofnormal telogen follicles into the next hair cycle.

“Preventing”, “prevention”, “preventative” or “prophylactic” refers tokeeping from occurring, or to hinder, defend from, or protect from theoccurrence of a condition, disease, disorder, or phenotype, including anabnormality or symptom. A mammal in need of prevention may be prone todevelop the condition.

The term “ameliorate” or “amelioration” refers to a decrease, reductionor elimination of a condition, disease, disorder, or phenotype,including an abnormality or symptom.

The term “therapeutically effective amount” refers to an amount of theMidkine family protein capable of reducing hair thinning, hair loss oralopecia in a mammal to a level which is beneficial to treat or preventhair thinning, hair loss or alopecia. A therapeutically effective amountmay be determined empirically and in a routine manner in relation totreating hair thinning, hair loss or alopecia.

Throughout this specification, unless specifically stated otherwise orthe context requires otherwise, reference to a single step, compositionof matter, group of steps or group of compositions of matter shall betaken to encompass one and a plurality (i.e. one or more) of thosesteps, compositions of matter, groups of steps or group of compositionsof matter.

Each example described herein is to be applied mutatis mutandis to eachand every other example unless specifically stated otherwise.

Each definition or clarifying term described herein shall be taken toapply mutatis mutandis to each and every example of the invention unlessthe context requires otherwise.

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications. The invention alsoincludes all of the steps, features, compositions and compounds referredto or indicated in this specification, individually or collectively, andany and all combinations or any two or more of said steps or features.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides photographic representations showing a fur growthpromoting effect of thrice-weekly administration for 15 days of topicalformulations comprising midkine (MK) in 40% glycerol (1 μg/ml) or 10 μlof pleiotrophin (PTN) in 40% glycerol (1 μg/ml) to different alopecicregions of alopecic Black 6 MK heterozygous knockout (+/−) mice at 1.5years of age. In the drawing, the circles in each row indicate thepositioning of dermal patches of MK (circle nearer the neck region) andPTN (circles nearer the tail) for each animal in the same row. Panel Ashows an animal on the day treatment was commenced. Panels B-L show miceat day 1, 4, 6, 8, 11, 13, 15, 18, 20, 22, and 25 respectively fromcommencement of treatment. Data indicate that animals treated with atopical formulation of MK or PTN show significant fur regrowth in thisanimal model, and that fur regrowth occurs more rapidly using MK thanPTN. After 15 days when treatment ceased (panel H), MK had an ongoingbeneficial effect to day 25. Fur regrowth is not entirely localized tothe treated region, since animals showed improved fur condition overtheir entire dorsal region following treatment.

FIG. 2 provides photographic representations showing the effect of aparenteral, i.e., subcutaneous, formulations comprising MK (1 μg/ml) or50 μl of PTN (1 μg/ml) to different alopecic regions of aged Black 6 MKhomozygous knockout (−/−) mice at 1.5 years of age for 15 days, followedby treatment with topical formulations described in the legend to FIG. 1for a further 10 days. In the drawing, the circles in each row indicatethe positioning of subcutaneous injections and dermal patches of MK(circle nearer the neck region) and PTN (circles nearer the tail) foreach animal in the same row. Panel A shows an animal on the daytreatment was commenced. Panels B-L show mice at day 1, 4, 6, 8, 11, 13,15, 18, 20, 22, and 25 respectively from commencement of treatment.Panel H shows an animal on the day that thrice-weekly topical treatmentcommenced. Data indicate that animals treated with a sub-cutaneousinjection of MK or PTN showed no macroscopic signs of fur regrowth 15days later. However, 10 days following commencement of topical therapy,administration of MK or PTN had stimulated fur growth in this animalmodel (panel L). Fur regrowth was not entirely localized to the treatedregion, since animals showed improved fur condition over their entiredorsal region following treatment.

FIG. 3 provides photographic representations showing a fur-growthpromoting effect of externally applying 100 μl of MK in 10% glycerol (2μg/ml) five times per week to alopecic regions of Black 6 MKheterozygous knockout (+/−) mice at 1.5 years of age. Panel A showsanimals on the day treatment was commenced. Panels B-F show mice at day7, day 14, day 21, day 28, and day 35 respectively from commencement oftreatment. Data show progressive fur regrowth during the 35 day testperiod, with significant fur regrowth evident after about 21-28 days(panels C and D).

FIG. 4 provides photographic representations showing a fur-growthpromoting effect of externally applying 100 μl of PTN in 10% glycerol (2μg/ml)five times per week to alopecic regions of Black 6 MK heterozygousknockout (+/−) mice at 1.5 years of age. Panel A shows animals on theday treatment was commenced. Panels B-F show mice at day 7, day 14, day21, day 28, and day 35 respectively from commencement of treatment. Datashow progressive fur regrowth during the 35 day test period, withsignificant fur regrowth evident after about 21-28 days (panels C andD).

FIG. 5 provides photographic representations showing the fur-growthpromoting effect of externally applying 100 μl of minoxidil (RiUP®,Taisho Pharmaceutical Co., Ltd.) to alopecic regions of aged Black 6 MKheterozygous knockout (+/−) mice as a positive control to theexperiments of FIGS. 3 and 4. Panel A shows animals on the day treatmentwas commenced. Panels B-F show mice at day 7, day 14, day 21, day 28,and day 35 respectively from commencement of treatment. Data show lesseffective treatment with minoxidil than that observed followingtreatment with MK or PTN because substantial areas of skin failed toregrow fur after 35 days treatment with minoxidil.

FIG. 6 provides photographic representations showing fur regrowth inshaved regions of nine-week old male C3H/HeJ mice following externalapplication of 100 μl of 10% (v/v) glycerol/phosphate buffered saline(PBS) as a negative control for the experiments of FIGS. 8 and 9. PanelA shows animals on the day treatment was commenced. Panels B-D show miceat day 7, day 14, and day 21 respectively from commencement oftreatment. Data show slow fur regrowth which becomes apparentmacroscopically only on about day 21.

FIG. 7 provides photographic representations showing fur regrowth inshaved regions of nine-week old male C3H/HeJ mice following externalapplication of 100 μl of minoxidil (e.g., 1% w/v; RiUP®, TaishoPharmaceutical Co., Ltd.) as a positive control for the experiments ofFIGS. 8 and 9. Panel A shows animals on the day treatment was commenced.Panels B-D show mice at day 7, day 14, and day 21 respectively fromcommencement of treatment. Data show slow fur re-growth which becomesapparent macroscopically only on about day 21, consistent with thevasodilatory effect of minoxidil on dermal papillae.

FIG. 8 provides photographic representations showing fur regrowth inshaved regions of nine-week old male C3H/HeJ mice following externalapplication of 100 μl of MK in 10% glycerol/PBS (2 μg/ml). Panel A showsanimals on the day treatment was commenced. Panels B-D show mice at day7, day 14, and day 21 respectively from commencement of treatment. Datashow slow fur regrowth which becomes apparent macroscopically only onabout day 21, suggesting a different mode of action for MK to minoxidilin animals that have merely been synchronized in the anagen phase,without exhibiting symptoms of alopecia.

FIG. 9 provides photographic representations showing fur regrowth inshaved regions of nine-week old male C3H/HeJ mice following externalapplication of 100 μl of PTN in 10% glycerol/PBS (2μg/ml). Panel A showsanimals on the day treatment was commenced. Panels B-D show mice at day7, day 14, and day 21 respectively from commencement of treatment. Datashow slow fur regrowth Which becomes apparent macroscopically only onabout day 21, suggesting a different mode of action for MK to minoxidilin animals that have merely been synchronized in the anagen phase,without exhibiting symptoms of alopecia

FIG. 10 provides a graphical representation showing the percentagealopecia developed in a murine model of cyclophosphamide-inducedalopecia over time (days) following synchronization in anagen phase byshaving and waxing on day 1, and 150 mg/kg cyclophosphamide (CYP)injection i/p on day 9. Percentage alopecia was determined as thepercentage of the area of skin to which the dermal patches were appliedshowing fur loss on the indicated day of measurement. Circles showalopecia in animals receiving daily saline placebo i.e., without midkineprotein, in addition to CYP. Filled squares (MK 2-29) show percentagealopecia in animals receiving 5 μg midkine protein topically on a dailybasis from day 2 to day 29 when the experiment was terminated. Filledtriangles (MK-10-29) show percentage alopecia in animals receiving 5 μgmidkine protein each on a daily basis from day 10 to day 29 when theexperiment was terminated. Data demonstrate that midkine protein reducesCYP-induced fur loss when administered before and/or following CYP, andthat midkine increases a rate of follicle recovery as determined by furgrowth following cessation of CYP treatment. Under these conditions,maximum differentiation between animals receiving the placebo controland animals receiving midkine was detectable on days 12-14.

FIG. 11 provides a graphical representation of the percentage alopeciaon day 13 of the data presented in FIG. 10. Percentage alopecia wasdetermined as the percentage of the area of skin to which the dermalpatches were applied showing fur loss on the indicated day ofmeasurement. The open bar (PBS) shows 42% alopecia in animals receivingdaily saline placebo i.e., without midkine protein, in addition to CYP.The hatched bar (MK 2-29) show only 20% alopecia in animals receiving 5μg midkine protein topically on a daily basis from day 2 to day 29 whenthe experiment was terminated. The striped bar (MK-10-29) shows only 26%alopecia in animals receiving 5 μg midkine protein each on a daily basisfrom day 10 to day 29 when the experiment was terminated. Datademonstrate that midkine protein reduces CYP-induced fur loss whenadministered before and/or following CYP, and that midkine increases arate of follicle recovery as determined by fur growth followingcessation of CYP treatment.

FIG. 12 provides photographic representations of the back skin of 15mice, each treated with phosphate buffered saline placebo i.e., withoutmidkine protein, administered daily as a topical formulation (group of 5mice labeled PBS), or 5 μg midkine protein administered daily as atopical formulation before and/or after receiving a singlecyclophosphamide (CYP) injection i/p (group of 5 mice labeled MK 2-29),or treated topically with 5 μg midkine protein on a daily basis afterreceiving a single 150 mg/kg cyclophosphamide (CYP) injection i/p (groupof 5 mice labeled MK 10-29). Animals were observed for signs of furregrowth/alopecia and skin pigmentation. Data indicate that mice treatedwith PBS exhibit extensive fur loss and grey or pink skin colour,especially in their neck regions, consistent with a predominance offollicles in catagen (grey) or telogen (pink). Mice treated with midkineprotein before and after CYP treatment (MK2-29) showed reduced areas offur loss relative to the group receiving PBS, and skin was eitheruniformly black or dark grey in color consistent with a predominance offollicles in anagen (black) and a lower proportion in catagen. Almost nopink skin was evident in this group, suggesting a much reducedproportion of follicles in telogen. Similarly, mice treated with midkineprotein only following CYP treatment (MK10-29) showed reduced areas offur loss relative to the group receiving PBS, and appearedmacroscopically similar to animals receiving midkine protein before andafter chemotherapy. The skin of mice receiving midkine protein onlyafter chemotherapy was uniformly dark grey in color, consistent with apredominance of follicles in anagen or catagen.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NO: 1 provides the amino acid sequence of human midkine(Accession identifiers gi|4505135 and ref|NP_(—)002382.1).

SEQ ID NO: 2 provides the amino acid sequence of human pleiotrophin(Accession identifiers gi|4506281 and ref|NP_(—)002816.1),

SEQ ID NO: 3 provides the amino acid sequence of a human midkine-likeprotein corresponding with SEQ ID NO: 4 of WO 2004/052928).

SEQ ID NO: 4 provides the nucleotide sequence of Human midkine mRNA(Accession identifiers gi|182650 and gb|M69148.1).

DETAILED DESCRIPTION OF THE INVENTION Midkine Family Protein

Midkine is a 13-kDa protein, which was discovered as a product from agene whose expression is induced in an early stage of lung tumour celldifferentiation due to retinoic acid. Pleiotrophin was discovered in thebrain of a newborn rat as heparin-binding protein with an ability ofenhancing neurite extension.

As used herein a “Midkine family protein” is a protein that exhibits asimilar amino acid sequence to midkine or a functional region thereofand exhibits the same function as or a similar function to that ofmidkine. A Midkine family protein includes a functional variant of aMidkine family protein.

Even a protein with low identity with midkine may be a Midkine familyprotein, provided that the protein has the same function as or a similarfunction to that of midkine. An example of such a Midkine family proteinmay be midkine (e.g. SEQ ID NO: 1), midkine-like protein (e.g. SEQ IDNO: 3 (WO 2004/052928)), truncated midkine protein, or pleiotrophin(e.g. SEQ ID NO: 2). The Midkine family protein may be a functionalvariant of a Midkine family protein, including a functional variant ofmidkine, midkine-like protein, truncated midkine protein, orpleiotrophin. The functional variant may be modified by substitution,deletion, or addition of one or more amino acids relative to thenon-modified protein. The functional variant of the Midkine familyprotein will exhibit a function of midkine.

In one embodiment, the Midkine family protein is midkine. In anotherembodiment, the Midkine family protein is pleiotrophin.

The function of midkine may be a function of enhancing cellproliferation, inhibiting apoptosis, binding to heparin, enhancing cellmigration, or inducing cell differentiation. In one embodiment, thefunction of MK is inhibiting apoptosis.

“Functional variant” and “variant” as used herein includes eithernatural protein variants or artificially modified protein variants thatexhibit a function of midkine.

As used herein, “modified” or “modification” includes substitution,addition, and/or deletion of an amino acid residue.

The Midkine family protein may have an amino acid sequence that is atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99% or is 100% identical over its length orfunctional region to a segment (preferably a continuous segment) of awild-type Midkine family protein (e.g. any one of SEQ ID NOs: 1 to 3).

A Midkine family protein may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10,or even 15, 20, 25, 30, 35, 40, 45, or 50 modifications e.g., atselected positions to maintain or enhance midkine function.

Thus, the specifically stated Midkine family protein sequences can vary,provided individual substitution, deletion and/or addition of an aminoacid does not strongly impair the function of the Midkine familyprotein.

The Midkine family protein or their functional variants can also belinked with other peptides or polypeptides or with further chemicalgroups such as glycosyl groups, lipids, phosphates, acetyl groups or thelike, provided they do not strongly adversely influence their midkinefunction. Thus, the modified Midkine family protein may be a fusionconstruct. For example, the Midkine family protei may be fused to apeptidyl moiety comprising an art-recognized protein translocationdomain to facilitate entry of the Midkine family protein to thefollicular cells.

In addition to naturally occurring amino acids, non-naturally occurringamino acids, or modified amino acids, are also contemplated and withinthe scope of the invention. In fact, as used herein, “amino acid” refersto naturally occurring amino acids, non-naturally occurring amino acids,and amino acid analogs, and to the D or L stereoisomers of each.

Natural amino acids include alanine (A), arginine (R), asparagine (N),aspartic acid (D), cysteine (C), glutamine (Q), glutamic acid (E),glycine (G), histidine (H), isoleucine (I), leucine (L), lysine (K),methionine (M), phenylalanine (F), proline (P), serine (S), threonine(T), tryptophan (W), tyrosine (Y), valine (V), hydroxyproline (O and/orHyp), isodityrosine (IDT), and di-isodityrosine (di-IDT).Hydroxyproline, isodityrosine, and di-isodityrosine are formedpost-translationally: In some embodiments, the natural amino acids, inparticular the 20 genetically encoded amino acids, are used.

The substitutions may be conservative amino acid substitutions, in whichthe substituted amino acid has similar structural or chemical propertieswith the corresponding amino acid in the reference sequence.Alternatively, the substitutions may be non-conservative amino acidsubstitutions.

By way of example, conservative amino acid substitutions involvesubstitution of one aliphatic or hydrophobic amino acids, e.g., alanine,valine, leucine and isoleucine, with another; substitution of onehydroxyl-containing amino acid, e.g., serine and threonine, withanother; substitution of one acidic residue, e.g., glutamic acid oraspartic acid, with another; replacement of one amide-containingresidue, e.g., asparagine and glutamine, with another; replacement ofone aromatic residue, e.g., phenylalanine and tyrosine, with another;replacement of one basic residue, e.g., lysine, arginine and histidine,with another; and replacement of one small amino acid, e.g., alanine,serine, threonine, methionine, and glycine, with another.

Functional variants may be obtained in which the Midkine family proteinhas been chemically modified at the level of amino acid side chains, ofamino acid chirality, and/or of the peptide backbone. These alterationsare intended to provide proteins having similar or improved therapeuticproperties.

Modifications providing increased potency, prolonged activity, ease ofpurification, and/or increased half-life will be known to the personskilled in the art.

Functional variants of the Midkine family protein may be identified bymodifying the sequence of the protein and then assaying the resultingprotein for the ability to function similarly to midkine, for exampleenhancing cell proliferation, inhibiting apoptosis, binding to heparin,enhancing cell migration, or inducing cell differentiation.

The Midkine family protein can be made by synthetic chemistry ofrecombinant DNA mutagenesis techniques that are well known to personsskilled in the art. For example, midkine may be produced in accordancewith a method disclosed in the Examples of JP-A-H09-95454.

Formulations

The Midkine family protein may be formulated in any form used in thepharmaceutical, quasi-drug, or cosmetic field, preferably suitable fortopical administration. For example, the composition may be ahair-growing product, hair or scalp cosmetic (e.g.

shampoo, hair conditioner, scalp lotion, scalp cream, hair tonic, etc.),skincare product (e.g. lotion, cream, face cream, face lotion, milk,pack, liquid facial wash, soap, etc.), body care product (e.g. bodycream, body lotion, soap, liquid wash, bath additive, etc.), UVprotective agent (e.g. sun block, sunscreen lotion, tanning oil, etc.),or cosmetic (e.g. eyeliner, eyebrow pencil, cream, lotion, etc).

Conveniently, the Midkine family protein may be formulated forparenteral administration e.g., with one or more chemotherapeutic drugs,such as by intravenous injection.

Excipients will typically be included in the dosage form e.g., toimprove solubility and/or bioadhesion. Suitable excipients includesolvents, co-solvents, emulsifiers, plasticizers, surfactants,thickeners, pH modifiers, emollients, antioxidants, and chelatingagents, wetting agents, and water absorbing agents. Formulations mayalso include one or more additives, for example, dyes, colored pigments,pearlescent agents, deodorizers, and odor maskers.

Diluents or fillers increase the bulk of a solid dosage form so that apractical size is provided for compression of tablets or formation ofbeads and granules. Suitable diluents include, but are not limited todicalcium phosphate dihydrate, calcium sulfate, lactose, sucrose,mannitol, sorbitol, cellulose, microcrystalline cellulose, kaolin,sodium chloride, dry starch, hydrolyzed starches, pregelatinized starch,silicone dioxide, titanium oxide, magnesium aluminum silicate andpowdered sugar.

Formulations may also comprise one or more dispersants e.g.,phosphate-buffered saline (PBS), saline, glucose, sodium lauryl sulfate(SLS), polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), andhydroxypropylmethylcellulose (HPMC).

Formulations may also comprise one or more binders to impart cohesivequalities to a solid dosage formulation, and thus ensure that a tablet,bead or granule remains intact after the formation of the dosage forms.Suitable binder materials include, but are not limited to, starch,pregelatinized starch, gelatin, sugars (including sucrose, glucose,dextrose, lactose and sorbitol), polyethylene glycol, waxes, natural andsynthetic gums such as acacia, tragacanth, sodium alginate, cellulose,including hydroxypropylmethylcellulose (“HPMC”), microcrystallinecellulose (“MCC”), hydroxypropylcellulose, ethylcellulose, and veegum,and synthetic polymers such as acrylic acid and methacrylic acidcopolymers, methacrylic acid copolymers, methyl methacrylate copolymers,aminoalkyl methacrylate copolymers, polyacrylic acid/polymethacrylicacid and polyvinylpyrrolidone (PVP).

Formulations may also comprise one or more lubricants to facilitatemanufacture or ingestion of a solid dosage unit e.g., a tablet. Examplesof suitable lubricants include, but are not limited to, magnesiumstearate, calcium stearate, stearic acid, glycerol behenate,polyethylene glycol, talc, and mineral oil.

Formulations may also comprise one or more disintegrants to facilitatedosage form disintegration after administration, and generally include,but are not limited to, starch, sodium starch glycolate, sodiumcarboxymethyl starch, sodium carboxymethylcellulose, hydroxypropylcellulose, pregelatinized starch, clays, cellulose, alginine, gums orcross linked polymers, such as cross-linked PVP.

Formulations may also comprise one or more stabilizers and/orpreservatives (e.g., E216, E218, and chlorobutanol hemihydrate) toinhibit or retard drug decomposition reactions e.g., by oxidation orbacterial action.

Formulations may also comprise one or more surfactants. Surfactants maybe anionic, cationic, amphoteric or nonionic surface active agents.Suitable anionic surfactants include, but are not limited to, thosecontaining carboxylate, sulfonate and sulfate ions. Examples of anionicsurfactants include sodium, potassium, ammonium of long chain alkylsulfonates and alkyl aryl sulfonates such as sodium dodecylbenzenesulfonate; dialkyl sodium sulfosuccinates, such as sodium dodecylbenzenesulfonate; dialkyl sodium sulfosuccinates, such as sodiumbis-(2-ethylthioxyl)-sulfosuccinate; and alkyl sulfates such as sodiumlauryl sulfate. Cationic surfactants include, but are not limited to,quaternary ammonium compounds such as benzalkonium chloride,benzethonium chloride, cetrimonium bromide, stearyl dimethylbenzylammonium chloride, polyoxyethylene and coconut amine. Examples ofnonionic surfactants include ethylene glycol monostearate, propyleneglycol myristate, glyceryl monostearate, glyceryl stearate,polyglyceryl-4-oleate, sorbitan acylate, sucrose acylate, PEG-150laurate, PEG-00 monolaurate, polyoxyethylene monolaurate, polysorbates,polyoxyethylene octylphenylether, PEG-1000 cetyl ether, polyoxyethylenetridecyl ether, polypropylene glycol butyl ether, steamylmonoisopropanolamide, and polyoxyethylene hydrogenated tallow amide.Examples of amphoteric surfactants include sodium N-dodecyl-β-alanine,sodium N-lauryl-β-iminodipropionate, myristoamphoacetate, lauryl betaineand lauryl sulfobetaine.

If desired, solid formulations e.g., tablets, beads, granules, orparticles may also contain an amount of a non-toxic auxiliary substancesuch as a wetting or emulsifying agent, dye, or pH buffering agent.

Pharmaceutical formulations may be presented in unit dose formscontaining a predetermined amount of active agent i.e., Midkine familyprotein, or a derivative or analog thereof, per unit dose. Theconcentration of active agent may vary depending upon whether or not theformulation is for prevention or therapy, the route of administration,half-life of the compound following administration by the selectedroute, and the age, weight and condition of the patient including e.g.,the severity of problem drinking being treated. For example a unit dosemay comprise about 1 μg to 10 ug, or 0.01 mg to 1000 mg, or 0.1 mg to250 mg, of Midkine family protein, or a derivative or analog thereof. Inanother example, Midkine family protein, or a derivative or analogthereof may be formulated such that the concentration of active agent isat least about 1% (w/w) or at least about 5% (w/w) or at least about 10%(w/w) or at least about 25% (w/w) based on the total weight of thepharmaceutical composition.

To prepare pharmaceutical formulations, one or more Midkine familyproteins is/are mixed with a pharmaceutically acceptable carrier orexcipient for example, by mixing with physiologically acceptablecarriers, excipients, or stabilizers in the form of, e.g., lyophilizedpowders, slurries, aqueous solutions, or suspensions (see, e.g.,Hardman, et al. (2001) Goodman and Gilman's The Pharmacological Basis ofTherapeutics, McGraw-Hill, New York, N.Y.; Gennaro (2000) Remington: TheScience and Practice of Pharmacy, Lippincott, Williams, and Wilkins, NewYork, N.Y.; Avis, et al. (eds.) (1993) Pharmaceutical Dosage Forms:Parenteral Medications, Marcel Dekker, NY; Lieberman, et al. (eds.)(1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY;Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: DisperseSystems, Marcel Dekker, NY; Weiner and Kotkoskie (2000) ExcipientToxicity and Safety, Marcel Dekker, Inc., New York, N.Y.).

The amount of the Midkine family protein in a formulation for treatmentof alopecia may vary depending on various parameters such as age, sex,and condition of the mammal being treated. For example, the Midkinefamily protein may be formulated to produce a final concentration of0.001 to 50% (w/w), 0.001 to 10% (w/w), 0.001 to 5% (w/w), or 0.001 to1% of the total weight of the medicament. The concentration of theMidkine family protein in a liquid formulation may be, for example,0.0001 μg/ml to 2 mg/ml, 0.001 μg/ml to 1 mg/ml, 0.01 μg/ml to 500μg/ml, 0.1 μg/ml to 100 μg/ml, 0.1 μg,/ml to 50 μg/ml, 0.1 μg/ml to 10μg/ml, or 1 μg/ml to 2 μg/ml.

A formulation comprising a midkine family protein and a pharmaceuticallyacceptable carrier or diluent may further comprise one or adjunctivetherapeutic agents for treatment of the same condition, wherein theadjunctive therapeutic agent is suitable for administration by the sameroute as the midkine family protein(s). For example, a midkine familyprotein may be formulated for topical administration with cestradioland/or oxandrolone and/or minoxidil. The adjunctive therapeutic agent isgenerally present in the formulation at a concentration in accordancewith its known prescribed level. The skilled artisan will appreciatethat such compositions may provide enhanced therapeutic benefit to thepatient, and may be more than additive in their effect.

Formulation of a pharmaceutical compound will vary according to theroute of administration selected (e.g., solution, emulsion, capsule).For solutions or emulsions, suitable carriers include, for example,,aqueous- or alcoholic/aqueous solutions, emulsions or suspensions,including saline and buffered media. Parenteral vehicles can includesodium chloride solution, Ringer's dextrose, dextrose and sodiumchloride, lactated Ringer's or fixed oils, for instance. Intravenousvehicles can include various additives, preservatives, or fluid,nutrient or electrolyte replenishers and the like (See, generally,Remington's Pharmaceutical Sciences, 17th Edition, Mack Publishing Co.,Pa., 1985). For inhalation, the agent can be solubilized and loaded intoa suitable dispenser for administration (e.g., an atomizer, nebulizer orpressurized aerosol dispenser).

Pharmaceutical formulations can be adapted for administration by anyappropriate route, for example by the oral (including buccal orsublingual), topical (including buccal, sublingual or transdermal), orparenteral (including subcutaneous, intramuscular, intravenous orintradermal) route. Such formulations can be prepared by any methodknown in the art of pharmacy, for example by bringing into associationthe active ingredient with the carrier(s), diluent(s) or excipient(s).

For example, the Midkine family protein is formulated for topicaladministration. Topical compositions include those pharmaceutical formsin which the Midkine family protein(s) is(are) applied externally bydirect contact with the skin surface to be treated. Conventionalpharmaceutical forms for this purpose include ointments, liniments,creams, shampoos, lotions, pastes, jellies, sprays, aerosols, and thelike, and may be applied in patches or impregnated dressings dependingon the part of the body to be treated. The term “ointment” embracesformulations (including creams) having oleaginous, water-soluble andemulsion-type bases, e.g., petrolatum, lanolin, polyethylene glycols, aswell as mixtures thereof.

For topical use on the eyelids or eyebrows, the active Midkine familyprotein(s) can be formulated in aqueous alcohol solutions, creams,ointments or oils exhibiting physiologically acceptable osmolarity byaddition of pharmacologically acceptable buffers and salts. Suchformulations may or may not, depending on the dispenser, containpreservatives such as benzalkonium chloride, chlorhexidine,chlorobutanol, parahydroxybenzoic acids and phenylmercuric salts such asnitrate, chloride, acetate, and borate, or antioxidants, as well asadditives like EDTA, sorbitol, boric acid etc. as additives.Furthermore, particularly aqueous solutions may contain viscosityincreasing agents such as polysaccharides, e.g., methylcellulose,mucopolysaccharides, e.g., hyaluronic acid and chondroitin sulfate, orpolyalcohol, e.g., polyvinylalcohol. Various slow releasing gels andmatrices may also be employed as well as soluble and insoluble ocularinserts, for instance, based on substances forming in-situ gels.Depending on the actual formulation and Midkine family protein(s) to beused, various amounts of the drug and different dose regimens may beemployed.

For topical use on the skin and the scalp, the Midkine family protein(s)can be advantageously formulated using ointments, creams, liniments orpatches as a carrier of the active ingredient. Also, these formulationsmay or may not contain preservatives, depending on the dispenser andnature of use. Such preservatives include those mentioned above, andmethyl-, propyl-, or butyl-parahydroxybenzoic acid, betain,chlorhexidine, benzalkonium chloride, and the like. Various matrices forslow release delivery may also be used.

In another example, a formulation comprising one or more Midkine familyproteins is adapted for parenteral administration e.g., by subcutaneousor intravenous injection. Such formulations include aqueous andnon-aqueous sterile injection solutions which may contain theantioxidants as well as buffers, bacteriostats and solutes that renderthe formulation isotonic with the blood of the intended recipient; andaqueous and non-aqueous sterile suspensions which may include suspendingagents and thickening agents. The formulations may be presented inunit-dose or multi-dose containers, for example sealed ampules andvials, and may be stored in a freeze-dried (lyophilized) conditionrequiring only the addition of the sterile liquid carrier, for examplewater for injections, immediately prior to use. Extemporaneous injectionsolutions and suspensions may be prepared from sterile powders, granulesand tablets. In one example, one or more Midkine family proteins isformulated as an intravenous lipid emulsion or a surfactant micelle orpolymeric micelle (see., e.g., Jones et al., Eur. J. PharmaceuticsBiopharmaceutics 48, 101-111, 1999; Torchilin J. Clin, release 73,137-172, 2001 for parenteral administration.

Sustained release injectable formulations are produced e.g., byencapsulating one or more Midkine family proteins in porousmicroparticles comprising a pharmaceutical agent and a matrix materialhaving a volume average diameter between about 1 μm and 150 μm, e.g.,between about 5 μm and 25 μm diameter. In one example; the porousmicroparticles have an average porosity between about 5% and 90% byvolume. In another example, the porous microparticles further compriseone or more surfactants, such as a phospholipid. The microparticles maybe dispersed in a pharmaceutically acceptable aqueous or non-aqueousvehicle for injection. Suitable matrix materials for such formulationscomprise a biocompatible synthetic polymer, a lipid, a hydrophobicmolecule, or a combination thereof. For example, the synthetic polymercan comprise, for example, a polymer selected from the group consistingof poly(hydroxy acids) such as poly(lactic acid), poly(glycolic acid),and poly(lactic acid-co-glycolic acid), poly(lactide), poly(glycolide),poly(lactide-co-glycolide), polyanhydrides, polyorthoesters, polyamides,polycarbonates, polyalkylenes such as polyethylene and polypropylene,polyalkylene glycols such as poly(ethylene glycol), polyalkylene oxidessuch as poly(ethylene oxide), polyalkylene terepthalates such aspoly(ethylene terephthalate), polyvinyl alcohols, polyvinyl ethers,polyvinyl esters, polyvinyl halides such as poly(vinyl chloride),polyvinylpyrrolidone, polysiloxanes, poly(vinyl alcohols), poly(vinylacetate), polystyrene, polyurethanes and co-polymers thereof,derivativized celluloses such as alkyl cellulose, hydroxyalkylcelluloses, cellulose ethers, cellulose esters, nitro celluloses, methylcellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxy-propylmethyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate,cellulose propionate, cellulose acetate butyrate, cellulose acetatephthalate, carboxylethyl cellulose, cellulose triacetate, and cellulosesulphate sodium salt (jointly referred to herein as “syntheticcelluloses”), polymers of acrylic acid, methacrylic acid or copolymersor derivatives thereof including esters, poly(methyl methacrylate),poly(ethyl methacrylate), poly(butylmethacrylate), poly(isobutylmethacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate),poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methylacrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), andpoly(octadecyl acrylate) (jointly referred to herein as “polyacrylicacids”), poly(butyric acid), poly(valeric acid), andpoly(lactide-co-caprolactone), copolymers, derivatives and blendsthereof. In a preferred example, the synthetic polymer comprises apoly(lactic acid), a poly(glycolic acid), a poly(lactic-co-glycolicacid), or a poly(lactide-co-glycolide).

In another example, pharmaceutical formulations are adapted for oraladministration e.g., as capsules, soft gels, or tablets; powders orgranules, solutions or suspensions in aqueous or non-aqueous liquids,edible foams or whips, or oil-in-water liquid emulsions or water-in-oilliquid emulsions. An oral formulation may comprise an intragranularphase comprising an effective amount of a Midkine family protein and atleast one carbohydrate alcohol and an aqueous binder. The pharmaceuticalformulation may be substantially lactose-free. Preferred carbohydratealcohols for such formulations are selected from the group consisting ofmannitol, maltitol, sorbitol, lactitol, erythritol and xylitol.Preferably, the carbohydrate alcohol is present at a concentration ofabout 15% to about 90%. A preferred aqueous binder is selected from thegroup consisting of hydroxypropyl cellulose, hydroxypropylmethylcellulose, carboxymethyl cellulose sodium, polyvinyl pyrrolidones,starches, gelatins and the like. A binder is generally present in therange of from about 1% to about 15% by weight. The intragranular phasecan also comprise one or more diluents, such as, for example, a diluentselected from the group consisting of microcrystalline cellulose,powdered cellulose, calcium phosphate-dibasic, calcium sulfate,dextrates, dextrins, alginates and dextrose excipients. Such diluentsare also present in the range of about 15% to about 90% by weight. Theintragranular phase can also comprise one or more disintegrants, suchas, for example, a disintegrant selected from the group consisting of alow substituted hydroxypropyl cellulose, carboxymethyl cellulose,calcium carboxymethylcellulose, sodium carboxymethyl cellulose, sodiumstarch glycollate, crospovidone, croscarmellose sodium, starch,crystalline cellulose, hydroxypropyl starch, and partiallypregelatinized starch. A disintegrant is generally present in the rangeof from about 5% to about 20% by weight. Such a formulation can alsocomprise one or more lubricants such as, for example, a lubricantselected from the group consisting of talc, magnesium stearate, stearicacid, hydrogenated vegetable oils, glyceryl behenate, polyethyleneglycols and derivatives thereof, sodium lauryl sulphate and sodiumstearyl fumarate. A lubricant is generally present in the range of fromabout 0.5% to about 5% by weight. Such formulations are made into atablet, capsule, or soft gel e.g., by a process comprising mixing aMidkine family protein and at least one carbohydrate alcohol to form adry blend, wet granulating the dry blend with an aqueous binder so as toobtain an intragranular phase, and further formulating the resultingintragranular phase so as to provide the formulation. Typically, tabletor capsules is prepared to contain an appropriate unit dosage e.g., from0.001 mg to 1000 mg.

Alternatively, a liquid or semi-solid pharmaceutical formulation fororal administration e.g., a hard gel or soft gel capsule comprising oneor more Midkine family proteins may be prepared. The formulation maycomprise a first carrier component and optional second carriercomponent, which carriers comprise, independently, one or more oflauroyl macrogel glycerides, caprylocaproyl macrogel glycerides,stearoyl macrogel glycerides, linoleoyl macrogel glycerides, oleoylmacrogel glycerides, polyalkylene glycol, polyethylene glycol,polypropylene glycol, polyoxyethylene-polyoxypropylene copolymer, fattyalcohol, polyoxyethylene fatty alcohol ether, fatty acid,polyethoxylated fatty acid ester, propylene glycol fatty acid ester,fatty ester, glycerides of fatty acid, polyoxyethylene-glycerol fattyester, polyoxypropylene-glycerol fatty ester, polyglycolized glycerides,polyglycerol fatty acid ester, sorbitan ester, polyethoxylated sorbitanester, polyethoxylated cholesterol, polyethoxylated castor oil,polyethoxylated sterol, lecithin, glycerol, sorbic acid, sorbitol, orpolyethoxylated vegetable oil.

The formulation may also comprise an emulsifying/solubilizing componentcomprising one or more of metallic alkyl sulfate, quaternary ammoniumcompounds, salts of fatty acids, sulfosuccinates, taurates, amino acids,lauroyl macrogol glycerides, caprylocaproyl macrogolglycerides, stearoylmacrogol glycerides, linoleoyl macrogol glycerides, oleoyl macrogolglycerides, polyalkylene glycol, polyethylene glycol, polypropyleneglycol, polyoxyethylene-polyoxypropylene copolymer, polyoxyethylenefatty alcohol ether, fatty acid, polyethoxylated fatty acid ester,propylene glycol fatty acid ester, polyoxyethylene-glycerol fatty ester,polyglycolized glycerides, polyglycerol fatty acid ester, sorbitanester, polyethoxylated sorbitan ester, polyethoxylated cholesterol,polyethoxylated castor oil, polyethoxylated sterol, lecithin, orpolyethoxylated vegetable oil.

The formulation may also comprise an anti-crystallization/solubilizingcomponent which, when present, generally comprises one or more ofmetallic alkyl sulfate, polyvinylpyrrolidone, lauroyl macrogolglycerides, caprylocaproyl macrogolglycerides, stearoyl macrogolglycerides, linoleoyl macrogol glycerides, oleoyl macrogol glycerides,polyalkylene glycol, polyethylene glycol, polypropylene glycol,polyoxyethylene-polyoxypropylene copolymer, fatty alcohol,polyoxyethylene fatty alcohol ether, fatty acid, polyethoxylated fattyacid ester, propylene glycol fatty acid ester, fatty ester, glyceridesof fatty acid, polyoxyethylene-glycerol fatty ester, polyglycolizedglycerides, polyglycerol fatty acid ester, sorbitan ester,polyethoxylated sorbitan ester, polyethoxylated cholesterol,polyethoxylated castor oil, polyethoxylated sterol, lecithin, orpolyethoxylated vegetable oil.

A Midkine family protein may be formulated with a hydrophobic polymer;preferably a bioadhesive polymer and optionally encapsulated in ordispersed throughout a microparticle or nanoparticle. The bioadhesivepolymer improves gastrointestinal retention via adherence of theformulation to the walls of the gastrointestinal tract. Suitablebioadhesive polymers include polylactic acid, polystyrene, poly(biscarboxy phenoxy propand-co-sebacic anhydride) (20:80) (poly (CCP:SA)),alginate (freshly prepared); and poly(fumaric anhydride-co-sebacicanhydride (20:80) (poly (FA:SA)), types A (containing sudan red dye) andB (undyed). Other high-adhesion polymers include p(FA:SA) (50:50) andnon-water-soluble polyacrylates and polyacrylamides. Preferredbioadhesive polymers are typically hydrophobic enough to benon-water-soluble, but contain a sufficient amount of exposed surfacecarboxyl groups to promote adhesion e.g., non-water-solublepolyacrylates and polymethacrylates; polymers of hydroxy acids, such aspolylactide and polyglycolide; polyanhydrides; polyorthoesters; blendscomprising these polymers; and copolymers comprising the monomers ofthese polymers. Preferred biopolymers are bioerodable, with preferredmolecular weights ranging from 1000 to 15,000 kDa, and most preferably2000 to 5000 Da. Polyanhydrides e.g., polyadipic anhydride (“p(AA)”),polyfumaric anhydride, polysebacic anhydride, polymaleic anhydride,polymalic anhydride, polyphthalic anhydride, polyisophthalic anhydride,polyaspartic anhydride, polyterephthalic anhydride, polyisophthalicanhydride, poly carboxyphenoxypropane anhydride and copolymers withother polyanhydrides at different mole ratios, are particularlypreferred. Blends of hydrophilic polymers and bioadhesive hydrophobicpolymers can also be employed. Suitable hydrophilic polymers includee.g., hydroxypropylmethylcellulose, hydroxypropylcellulose,carboxymethylcellulose, polyvinylalcohols, polyvinylpyrollidones, andpolyethylene glycols. Other mucoadhesive polymers include DOPA-maleicanhydride co polymer, isopthalic anhydride polymer, DOPA-methacrylatepolymers, DOPA-cellulosic based polymers, and DOPA-acrylic acidpolymers.

Alternatively, the Midkine family protein may be encapsulated ormolecularly dispersed for oral administration in a polymer to reduceparticle size and increase dissolution. The polymers may includepolyesters such as poly(lactic acid) or P(LA), polycaprylactone,polylactide-coglycolide or P(LGA), poly hydroxybutyrate poly β-malicacid); polyanhydrides such as poly(adipic)anhydride or P(AA),poly(fumaric-co-sebacic)anhydride or P(FA:SA), poly(sebacic)anhydride orP(SA); cellulosic polymers such as ethylcellulose, cellulose acetate,cellulose acetate phthalate, etc; acrylate and methacrylate polymerssuch as Eudragit RS 100, RL 100, E100 PO, L100-55, L100, S100(distributed by Rohm America) or other polymers commonly used forencapsulation for pharmaceutical purposes and known to those skilled inthe art. Also suitable are hydrophobic polymers such as polyimides.Blending or copolymerization sufficient to provide a certain amount ofhydrophilic character can be useful to improve wetability of thematerials. For example, about 5% to about 20% of monomers may behydrophilic monomers. Hydrophilic polymers such ashydroxylpropylcellulose (HPC), hydroxpropylmethylcellulose (HPMC),carboxymethylcellulose (CMC) are commonly used for this purpose.

Oral formulations may be “immediate release” formulations e.g., thatrelease at least 85% (wt/wt) of the Midkine family protein within 60minutes in vitro. Alternatively, the formulation may be a “controlledrelease” formulation that releases drug more slowly than an immediaterelease formulation i.e., it takes longer than 60 minutes to release atleast 85% (wt/wt) of the drug in vitro. To extend the time period forrelease, the ratio of active agent to polymer can be increased.Increased relative drug concentration is believed to have the effect ofincreasing the effective compound domain size within the polymer matrixthereby slowing dissolution. In the case of a polymer matrix containingcertain types of hydrophobic polymers, the polymer will act as amucoadhesive material and increase the retention time of the activecompound in the gastrointestinal tract. Increased drug dissolution ratescombined with the mucoadhesive properties of the polymer matrix increaseuptake of the active compound and reduce differences found in the fedand fasted states for the compounds.

The compositions described herein may further comprise components whichare generally used in cosmetics, for example, oils, detergents, UVabsorbers, alcohols, chelating agents, pH modifiers, preservatives,thickeners, pigments, fragrances, and skin nutritional supplements.Specifically, the composition may comprise active ingredients used forskin cosmetics, such as zinc oxide microparticles, titanium oxide, UVabsorbers such as Parsol MCX and Parsol 1789, vitamins such as ascorbicacid, moisturising agents such as hyaluronate sodium, petrolatum,glycerin, and urea, hormonal agents, skin-lightening agents such askojic acid, arbutin, placenta extract, and rucinol, steroid drugs,inhibitors of production or release of a chemical mediator such asarachidonate metabolite and histamine (e.g. indometacin and ibuprofen),anti-inflammatory drugs such as receptor antagonist, anti-androgenicagents, sebum secretion suppressing agents such as vitamin A acid, royaljelly extract, and royal jelly acid, peripheral blood-vessel dilatorssuch as tocopherol nicotinate, alprostadil, isoxsuprine hydrochloride,and tolazoline hydrochloride, carbon dioxide with peripheralblood-vessel dilating activity, blood circulation promoting agents suchas minoxidil, carpronium chloride, capsicum tincture, vitamin Evariants, ginkgo extract, and Swertia japonica extract, cellularstimulants such as pentadecanoic acid glyceride and nicotinic-aid amide,antimicrobials such as hinokitiol, L-menthol, and isopropylmethylphenol,glycyrrhizipic acid and variants or salts thereof, ceramide and ceramideanalogs.

Dosage Units and Frequency of Administration

The dose and frequency of the Midkine family protein or the medicamentmay be appropriately modified depending on the situation.

In general, the Midkine family protein, or the medicament comprising theMidkine family protein(s), may be used with any frequency. Typically,the Midkine family protein(s) are applied repeatedly for a sustainedperiod of time topically on the part of the body to be treated, forexample, the eyelids, eyebrows, skin or scalp. The preferred dosageregimen will generally involve regular, such as daily, weekly,twice-weekly, or thrice-weekly, administration for a period of treatmentof at least one about one month, more preferably at least three months,and most preferably at least six months. For example, the Midkine familyprotein or the medicament may be used 1, 2, 3, 4, 5, 6 or 7 times perweek, corresponding with one use per day that the Midkine family proteinor medicament is applied. On any day, use of the Midkine family proteinor medicament may correspond with application 1, 2, 3, 4 or 5 times perday. In one embodiment, the Midkine family protein or medicament isapplied to the mammal once per day and three or five times per week.

Typically, a topical formulation comprising a Midkine family protein maybe applied in the amount of 0.001 to 1,000 μg/cm²/day, 0.005 to 500μg/cm²/day, 0.01 to 100 μg/cm²/day, 0.05 to 50 μg/cm²/day, or 0.1 to 10μg/cm²/day. Typically, the dose to be applied topically on the scalp isin the range of about 0.1 ng to about 100 mg per day, more preferablyabout 1 ng to about 10 mg per day, and most preferably about 10 ng toabout 1 mg per day depending on the Midkine family protein(s) and theformulation.

Typically, the daily amount of Midkine family protein(s) for treatmentof the eyelid may be about 0.1 ng to about 100 mg per eyelid.

The Midkine family protein may be applied alone as an active ingredient,or may be applied with other active ingredients. Similarly, themedicament may comprise further active ingredients in addition to theMidkine family protein. Such active ingredients other than the Midkinefamily protein may be, but are not limited to, cellular stimulants,blood circulation promoting agents, anti-androgen drugs, sebum secretionsuppressing agents, immunosuppressants, antihistamine agents,antimicrobials, focal stimulants, emollients, antiphlogistics, orlow-molecular anti-apoptotic agents. Specifically, said other activeingredients may be at least one of pantothenic acid or variants thereof,placenta extract, photosensitizers, ginseng extract, biotin, mononitroguaiacol, carpronium chloride or hydrates thereof, vitamin E or variantsthereof, Swertia japonica extract, capsicum tincture, cepharanthine,nicotinic acid or variants thereof, estradiol, ethynylestradiol, randicacid, minoxidil or analogs/variants thereof, 5α-reductase inhibitor,12-tetradecanoylphorbol-13-acetate, herbal medicine such as Polygonatumrhizome, Uncaria, Silybum marianum, henna, and Glycyrrhiza, estradiolbenzoate, diphenhydramine, resorcin, hinokitiol, 1-menthol, salicylicacid, Polygonum root extract, Panax japonicus rhizome extract,panthenol, selenium disulfide, pyridoxine hydrochloride, dipyrithionezinc, pyrithione zinc, sulfur, piroctone olamine, pyrithione zinc,sulfur, glycyrrhetinic acid stearyl, glycyrrhizinate dipotassium,allantoin, dialkylmonoamine variants, Perilla frutescens extract, Poriasclerotium extract, β-glycyrrhetinic acid, miconazole nitrate, benzoicacid, sodium salicylate, phytosterol, wine yeast extract, takanal,ethinyl estradiol, isopropylmethylphenol, cepharanthine biotin,D-pantothenyl alcohol, Paeonia extract, Tilia extract, Sophora extract,Sophora flavescens extract, Zingiber Officinale (Ginger) root extract,6-benzylaminoprine, pentadecanoic glyceride, t-flavanone, sweetHydrangea leaf extract, adenosine, and pantothenylethylether.

In one example, a formulation comprising a midkine family protein isadministered sequentially or simultaneously with an adjunctivetherapeutic agent for treatment of the same condition e.g., cestradioland/or oxandrolone and/or minoxidil and/or finasteride or an agent thatblocks the conversion of testosterone to dihydrotesterone. Theadjunctive therapeutic agent is co-administered under conditions and inaccordance to a standard treatment regime for that agent. The skilledartisan will appreciate that such treatment regimens provide enhancedtherapeutic benefit to the patient, and may be more than additive intheir effect.

Alternatively, or in addition, a formulation comprising a midkine familyprotein is administered sequentially or simultaneously with a cytotoxicor cytostatic compound that causes hair loss e.g., in the case of asubject undergoing chemotherapy or radiation therapy or treatment forHIV-1 infection or AIDS. In such circumstances, the efficacy of themidkine family protein counteracts the hair-loss effect of the cytotoxicor cytostatic compound. The cytotoxic or cytostatic compound willgenerally be administered in accordance to a standard treatment regimefor that agent. Conveniently, the Midkine family protein and thecytotxin/cytostatin are administered via the same route e.g.,parenterally.

Subjects

The compositions of the present invention are suitable for medicaltreatment of humans and other mammals, including treatment companionanimals such as dogs and cats, and domestic animals such as horses, zooanimals such as felids, canids, bovids, ungulates and primates, orlaboratory animals such as rodents, lagomorphs and primates. Thecompositions are particularly suitable for treatment of any mammal thatsuffers alopecia of any form, especially humans, primates dogs, cats, orhorses.

The subject to be treated may be afflicted with hair thinning, hair lossor alopecia, or may not be afflicted with hair thinning, hair loss oralopecia (i.e., free of detectable disease), but is prone to develophair thinning, hair loss or alopecia.

A Midkine family protein, or a medicament comprising a Midkine familyprotein, may be used to treat hair thinning, hair loss or alopeciacaused by cytotoxic or cytostatic agents. The cytotoxic or cytostaticagents may be endogenous, e.g. as generated in response to stress, ormay be exogenous, e.g. as administered during chemotherapy for treatmentof cancer.

The present invention is particularly suited to treatment and preventionof alopecia in subjects that are either undergoing treatment with acytotoxic or cytostatic compound described herein, or to whom suchtherapy has been prescribed. As exemplified herein, the subject may betreated before therapy with a cytotoxic or cytostatic compoundcommences, or before and after such therapy has commenced. The presentinvention also provides for commences of midkine family protein therapyafter treatment with a cytotoxic or cytostatic compound has commenced.

For example, a subject may apply a composition described herein as afine line at the skin-eyelash border of each eyelid, and as a cream tothe scalp, once a day several weeks e.g., two weeks or three weeks,prior to the initiation of a chemotherapy regimen (e.g., doxorubicin,cyclophosphamide, and paclitaxel, or 5-fluoruracil, leucovorin andoxaliplatin). The patient may continue applying the compositionthroughout and after cessation of the chemotherapy regimen. The patientwould not generally experience the total hair loss normally associatedwith chemotherapy, and may recover more rapidly when chemotherapyceases. A few weeks after completion of the chemotherapy, the patientmay stop applying the composition. If hair is lost at this stage,treatment is resumed.

The present invention is illustrated in detail below with references toexamples, but is not to be construed as being limited thereto.

Example 1 Method for Producing Recombinant Midkine

A cDNA fragment comprising human MK open reading frame (nucleotidepositions 1-432; SEQ ID NO: 4) was inserted into the yeast expressionvector pPIC9 (Invitrogen). This recombinant plasmid was transfected intoyeast (Pichia pastoris GS115; Research Corporation Technologies), andthe desired clones were selected with histidine and G418.

Human MK protein secreted by yeast into the culture medium was purifiedby employing the following column chromatography in the order below:

-   -   1. SP Steamlines (Pharmacia; adsorption and wash with 20 mM pH        5.5 acetate buffer, elution with 20 mM pH 3.5 acetate suffer        containing NaCl);    -   2. Sulfated Cellulofine (Seikagaku Kogyo, Japan; adsorption with        10 mM pH 7.2 phosphate buffer, wash with buffer containing 0.7 M        NaCl, elution with buffer containing 2.0 M NaCl);    -   3. Superdex 75 pg (Pharmacia; gel filtration with saline);    -   4. Poly. Sulfoethyl A (Poly LC Co.; adsorption with 20 mM buffer        containing 0.6 M NaCl, wash with buffer containing 0.88 M NaCl,        elution with buffer containing 2M NaCl); and    -   5. Superdex 75 pg (Pharmacia; gel filtration with saline).

The Midkine preparation was dialyzed against saline. The activity ofpurified MK protein was detected using as an index the activity of MKfor promoting the survival of embryonic neurons.

Example 2

Hair-growing effects of external applications of midkine (MK) andpleiotrophin (PTN). 1 μg/ml of MK or PTN in 40% glycerol/phosphatebuffered saline (PBS) (v/v) was prepared. 10 μl of the solution of MK orPTN was directly applied three times per week to alopecic regions ofaged Black 6 mice (1.5 years old, MK heterozygous knockout (+/−)). Itwas observed for 25 days after starting the application whether each ofMK and PTN had a hair-growing effect. FIG. 1 shows the results of theobservation. As clearly understood from FIG. 1 and the brief descriptionthereof, application of MK or PTN promoted hair growth on the alopecicregions of the aged mice. In this experiment, the applications of MK andPTN stopped on Day 15, because the hair-growing effect of MK and PTN wasalready confirmed on Day 15. From Day 15 to Day 25, the amount of hairincreased even though the applications of MK and PTN were stopped (FIG.1). This shows that the hair-growing effects of MK and PTN last for sometime even after their applications are stopped.

Example 3 Hair-Growing Effects of Subcutaneous Injection of MK and PTN

1 μg/ml of MK or PTN in PBS was prepared. 50 μl of the solution of MK orPTN was injected subcutaneously into aged Black 6 mice (1.5 years old,MK homozygous knockout (−/−)). The subcutaneous injections of MK and PTNshowed no hair-growing effect on Day 15 (FIG. 2) under these conditionsin this animal model. After changing on Day 15 from the subcutaneousinjection to external applications of MK or PTN in accordance withExample 2, the hair-growing effects of MK or PTN were observed on Day 25(FIG. 2).

Example 4 Hair-Growing Effect of Intravenous Injection of MK

1 μg/ml of MK in PBS was prepared. 100 μl of the solution of MK wasinjected intravenously into aged Black 6 mice (1.5 years old, MKhomozygous knockout (−/−)). The intravenous injection of MK showed nohair-growing effect under these conditions and in this animal model(data not shown).

Example 5 Hair-Growing Effects of External Applications of MK and PTN

2 μg/ml of MK or PTN in 10% glycerol/PBS (v/v) was prepared. 100 μl ofthe solution of MK or PTN was applied directly five times per week toalopecic regions of aged Black 6 mice (1.5 years old, MK heterozygousknockout (+/−)). It was observed for 35 days after starting theapplication whether each of MK and PTN had a hair-growing effect. FIGS.3 and 4 show the results of the observation. Minoxidil (e.g., 1% w/v;RiUP®, Taisho Pharmaceutical Co., Ltd.) was used as a positive control(FIG. 5). As clearly understood from FIGS. 3 and 4 and the briefdescriptions thereof, both applications of MK and PTN under theseconditions showed hair-growing effects on the alopecic regions of theaged mice.

Example 6 Hair-Growing Effects of External Applications of MK and PTNare Different to the Effect of Minoxidil

2 μg/ml of MK or PTN in 10% glycerol/PBS (v/v) was prepared. 100 μl ofthe solution of MK or PTN was applied directly to shaved regions ofC3H/HeJ mice (male, nine weeks old). 10% glycerol/PBS (v/v) andminoxidil (e.g., 1% w/v; RiUP®, Taisho Pharmaceutical Co., Ltd.) wereused as negative and positive controls, respectively (FIGS. 6 and 7). Itwas observed for 21 days after starting the application whether each ofMK and PTN showed enhancement of hair growth in the anagen phase. FIGS.8 and 9 show the results of the observation. The application ofminoxidil (RiUP®) showed enhancement of hair growth (FIG. 7). Incontrast, MK and PTN showed no such enhancement (FIGS. 8 and 9). Theavailable data suggest a different mode of action for MK to minoxidil inanimals that have merely been synchronized in the anagen phase, withoutexhibiting symptoms of alopecia.

Example 7 Effect of External Application of MK on Acute Alopecia AnimalEthics

All procedures were approved by the Royal Melbourne Institute ofTechnology (RMIT) Animal Experimentation and Ethics Committee (AEECProject #1042), Melbourne, Australia.

Animals

A total of thirty-five mice were used in the study.

Pathogen-free female C57B1/6J mice were obtained from Animal ResourceCentre, Canning Vale, Western Australia, Australia. Animals were housedunder standard clean conventional animal house conditions in a singleroom within the RDDT Animal Facility (201.01), maintained at targetvalueS of 22±2° C. and 30-70% relative humidity, with a 12 h light:darkcycle. Animals were fed irradiated Rat and Mouse Chow (Specialty Feeds,Glen Forrest, Western Australia, Australia) and supplied municipal townwater ad libitum. Fresh water was supplied at least twice weekly.Periodic testing of feed, bedding and water was undertaken to identifyany contaminants that might interfere with the study results.

Animals were randomised into study groups, housed in groups of two tothree per cage and subjected to at least a two-day quarantine period onreceipt. The animals were identified by implantation of microchiptransponders by sub-cutaneous injection between the shoulder blades.Secondary identification of animals within each cage was made by use oftail markings with a permanent marker. Mice were housed individuallyprior to the commencement of treatment if there appeared to be excessivegrooming of depilated mouse skin by cage-mates.

As the commencement of treatment, mice were aged between five weeks andseven weeks, and varied in weight by no more than 20% of their meanweight.

Induction of Acute Alopecia

Acute alopecia was induced according to the procedures described by Pauset al., Am. J. Pathol. 144, 719-734 (1994), i.e., by a singleintraperitoneal injection of cyclophosphamide to C57 BL/6 mice. Thestudy includes 35 female C57B1/6J mice divided into seven experimentalgroups of five mice each as outlined in the text table below:

Cyclophosphamide Midkine Treatment No. of Group mg/kg Study Day μg/mLStudy Day animals 1 0 9 0 2-29 5 2 50 9 0 2-29 5 3 50 9 10 2-29 5 4 50 910 10-29  5 5 150 9 0 2-29 5 6 150 9 10 2-29 5 7 150 9 10 10-29  5

The following protocol was employed:

Study Day 1: Mice were anaesthetized by intraperitoneal injection with acombination of ketamine (50 mg/kg), xylazine (5 mg/kg) and acepromazine(0.8 mg/kg). Fur was removed from a test area of approximately 2×4 cm onthe backs of the mice, by shaving with an electric clipper. The skin wasrinsed with warm water to clean the area and patted dry. The remainingfur was removed by use of a commercial hair remover wax strip.

Study Days,2-9: Mice in groups 1, 2, 3, 5 and 6 were administered afinal dose concentration of 5μg midkine/mouse/day, or a controlphosphate buffered saline (PBS) solution, by dermal application to thetest area in a volume of 0.5 mL. Mice in groups 4 and 7 wereadministered a control phosphate buffered saline (PBS) solution, bydermal application to the test area in a volume of 0.5 mL.

Study Day 9: Mice in all groups were treated with either vehicle (water)or cyclophosphamide by intraperitoneal injection in a dose volume of 10mL/kg.

Study Days 10-29: Mice in groups 4 and 7 were administered a final doseconcentration of 5 μg midkine/mouse/day, by dermal application to thetest area in a volume of 0.5 mL. Mice in groups 1, 2, 3, 5 and 6 wereadministered a final dose concentration of 5μg midkine/mouse/day, or acontrol phosphate buffered saline (PBS) solution, by dermal applicationto the test area in a volume of 0.5 mL. Animals in groups 1, 2, 3, 5,and 6 that received the control solution on days 2-9 also received thecontrol solution on days 10-29. Animals in groups 1, 2, 3, 5, and 6 thatreceived the midkine solution on days 2-9 also received the midkinesolution on days 10-29.

Study Day 30: Mice were sacrificed by exposure to a rising concentrationof carbon dioxide. A portion of skin in the target area was collectedand preserved in 10% neutral buffered formalin for macroscopic andmicroscopic examination.

Mice were monitored daily from Study Day 1-29 for clinical signs oftoxicity. These observations will include assessment of any changes inthe following:

-   -   Skin and fur (roughness, piloerection, lack of grooming, fur        loss);    -   Eyes and mucous membranes (discharge, cloudiness, sores around        eyes);    -   Respiratory, circulatory, autonomic or central nervous system        functions;    -   Somatomotor activity and behaviour patterns (check for abnormal        posture, gait and any abnormal activity);

Any tremors, convulsions, salivation, diarrhoea, lethargy, excessivesleeping or coma.

Skin/Fur Observations

From Study Day 9-29 onwards, mice were observed daily for signs ofalopecia (fur loss) and changes in skin colour pigmentation. The degreeof fur loss was determined as a percentage of target area with alopecia(fur loss), and skin pigmentation was scored as black, grey or pink.Group incidence findings for alopecia and skin pigmentation changes weredetermined for control and test groups by One-Way Analysis of Variancetests using GraphPad Prism 5.0 for Windows, GraphPad Software, San DiegoCalif. USA.

The extent of fur loss/re-growth was also documented by twice-weeklyphotographs. To facilitate the photography process, the mice weresedated using ketamine (50 mg/kg), xylazine (5 mg/kg), acepromazine (0.8mg/kg) anaesthetic.

Terminal Measurements

Skin samples were collected from the target area of each animal andpreserved in 10% neutral buffered formalin for histopathologicalassessment.

Data in FIG. 11 demonstrate that midkine protein reducescyclophosphamide-induced fur loss when administered before and/orfollowing cyclophosphamide, and that midkine increases a rate offollicle recovery as determined by fur growth following cessation ofcyclophosphamide treatment. Data in FIG. 12 indicate that mice treatedwith PBS exhibit extensive fur loss and grey or pink skin colour,especially in their neck regions, consistent with a predominance offollicles in catagen (grey) or telogen (pink). Mice treated with midkineprotein before and after CYP treatment (MK2-29) showed reduced areas offur loss relative to the group receiving PBS, and skin was eitheruniformly black or dark grey in color consistent with a predominance offollicles in anagen (black) and a lower proportion in catagen. Almost nopink skin was evident in this group, suggesting a much reducedproportion of follicles in telogen. Similarly, mice treated with midkineprotein only following CYP treatment (MK10-29) showed reduced areas offur loss relative to the group receiving PBS, and appearedmacroscopically similar to. animals receiving midkine protein before andafter chemotherapy. The skin of mice receiving midkine protein onlyafter chemotherapy was uniformly dark grey in color, consistent with apredominance of follicles in anagen or catagen.

1. A topical formulation comprising an amount of a midkine familyprotein and a topical carrier, excipient or emollient effective to treator prevent hair loss and/or promote hair growth and/or enhance hairgrowth.
 2. The topical formulation of claim 1 wherein said formulationis for administration to the dermis or skin of a subject who issusceptible to hair loss or at risk of hair loss.
 3. The topicalformulation of claim 1 wherein said formulation is for administration tothe dermis or skin of a subject suffering from hair loss.
 4. The topicalformulation according to claim 1 for treatment or prevention ofalopecia.
 5. The topical formulation according to claim 4, wherein thealopecia is an acute form of alopecia.
 6. The topical formulationaccording to claim 4 wherein the formulation is for treatment orprevention of alopecia in a subject undergoing treatment with acytotoxic agent or cytostatic agent or to whom treatment with acytotoxic agent or cytostatic agent has been prescribed.
 7. The topicalformulation according to claim 4, wherein the alopecia is androgenicalopecia.
 8. The topical formulation according to claim 1, wherein themidkine family protein is midkine, pleiotrophin, midkine-like protein,or a truncated midkine protein.
 9. The topical formulation according toclaim 1, wherein the midkine family protein is pleiotrophin.
 10. Thetopical formulation according to claim 1, wherein the midkine familyprotein is midkine.
 11. (canceled)
 12. (canceled)
 13. (canceled) 14.(canceled)
 15. (canceled)
 16. (canceled)
 17. (canceled)
 18. (canceled)19. A method of treatment or prevention of hair loss, said methodcomprising administering to a subject in need thereof a formulationcomprising an amount of a midkine family protein and a carrier,excipient or emollient, effective to prevent hair loss and/or promotehair growth and/or enhance hair growth on the subject.
 20. The methodaccording to claim 19, wherein the formulation is a topical formulationand wherein said method comprises administering the topical formulationto an affected area of the skin of the subject in which hair has beenlost or to an area of skin from which hair is likely to be lost.
 21. Themethod according to claim 19, wherein the method comprises administeringthe formulation to a subject suffering from alopecia for a time andunder conditions sufficient to reduce hair loss and/or effect hairgrowth in the subject.
 22. The method according to claim 19, wherein themethod comprises administering the formulation to a subject sufferingfrom alopecia for a time and under conditions sufficient to prevent orreduce apoptosis of follicles of the subject.
 23. The method accordingto claim 19, wherein the method comprises administering the formulationto a subject suffering from alopecia for a time and under conditionssufficient to extend an anagen phase of follicles of the subject. 24.The method according to claim 19, wherein the method comprisesadministering the formulation to a subject suffering from alopecia for atime and under conditions sufficient to promote or advance entry ofnormal telogen follicles of the subject into a following hair cycle. 25.The method according to claim 19, wherein the method comprisesadministering the formulation to a subject that is suffering fromalopecia.
 26. The method according to claim 1, wherein the methodcomprises administering the formulation to a subject that is susceptibleto developing alopecia.
 27. The method according to claim 25, whereinthe alopecia is an acute form of alopecia.
 28. The method according toclaim 27, wherein the method comprises administering the formulation toa subject undergoing treatment with a cytotoxic agent or cytostaticagent or to whom treatment with a cytotoxic agent or cytostatic agenthas been prescribed.
 29. The method according to claim 25, wherein thealopecia is androgenic alopecia.
 30. The method according to claim 19,wherein the midkine family protein is midkine, pleiotrophin,midkine-like protein, or a truncated midkine protein.
 31. The methodaccording to claim 19, wherein the midkine family protein ispleiotrophin.
 32. The method according to claim 19, wherein the midkinefamily protein is midkine.
 33. A method of promoting or enhancing hairgrowth or hair initiation in a subject suffering from hair loss, saidmethod comprising administering to the subject a topical formulationcomprising an amount of a midkine family protein and a topical carrier,excipient or emollient effective to prevent hair loss and/or promotehair growth and/or enhance hair growth on the subject.
 34. The methodaccording to claim 33, wherein the midkine family protein is midkine orpleiotrophin.
 35. A method of reducing hair loss in a subject undergoingchemotherapy or to whom chemotherapy has been prescribed, said methodcomprising administering to the subject a formulation comprising anamount of a midkine family protein and a carrier, excipient or emollientfor a time and under conditions sufficient to prevent or reduce hairloss due to the chemotherapy.
 36. The method according to claim 35,wherein the method comprises administering the formulation topically toa subject that has been treated with the chemotherapy.
 37. The methodaccording to claim 35, wherein the method comprises administering theformulation topically to a subject to whom chemotherapy has beenprescribed before commencement of the chemotherapy.
 38. The methodaccording to claim 35, wherein the method comprises administering theformulation topically to a subject to whom chemotherapy has beenprescribed before and after commencement of the chemotherapy.
 39. Themethod according to claim 35, wherein the midkine family protein ismidkine.
 40. A method of identifying or isolating a compound thatprevents hair loss and/or promotes hair growth and/or enhances hairgrowth, wherein said method comprises administering a test compound to amouse, said mouse being aged more than one year and suffering hair lossand having a knockout of one allele of a gene encoding a midkine proteinand one other functional allele of the gene, and then determining hairgrowth and/or reduced hair loss on the mouse, and wherein reduced hairloss and/or increased hair growth and/or an initiation of hair growth onthe mouse in the presence of the test compound relative to in theabsence of the test compound is indicative of the test compoundpreventing hair loss and/or promoting hair growth and/or enhancing hairgrowth.